Detection methods based on sequencing

ABSTRACT

Provided herein is a multi-sample and multi-locus method for analyzing a genetic locus. In particular, provided herein is a method for SNP detection and analysis based on high-throughput sequencing, comprising designing a probe, pre-amplification and biotin labeling, hybridization, ligation, barcode specific primer extension, sequencing and analyzing the SNP locus. A probe set is for the analysis is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase of International PatentApplication No. PCT/CN2015/000784, filed Nov. 10, 2015, which claimspriority to Chinese Patent Application No. 201410643497.9, filed on Nov.10, 2014, published as CN 104372093 A on Feb. 25, 2015, and the contentof each application is incorporated by reference herein in its entiretyfor all purposes.

Sequence Listing on ASCII Text

This patent or application file contains a Sequence Listing submitted incomputer readable ASCII text format (file name:4565-2008900_SeqList.txt; date recorded: Jul. 12, 2018; and size: 7,000bytes). The content of the Sequence Listing file is incorporated hereinby reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to the field of biotechnology, inparticular to an SNP or mutation detection method based on sequencingtechnology, such as high-throughput sequencing or second generatingsequencing.

BACKGROUND

SNP has extremely important value in molecular diagnostics, clinicaltesting, pathogen detection, forensics, genetic disease research,development of individual therapy and drugs, and many other fields(Gayet-Ageron et al., 2009). SNP detection is one of the main contentsof the current genetic diagnosis. At the same time, genetic diagnosisrepresented by SNP detection has become an important means of screeningnewborns or specific populations for genetic diseases. Therefore, aneasy-to-operate, low-cost and high-throughput SNP detection method iskey to genetic testing.

The second generation of high-throughput sequencing technology is moreaccurate, sensitive, with higher throughput compared to otherhigh-throughput gene detection technology. It has been involved invarious aspects of life science and medical research with its lowerprices and expanding range of applications. The use of high-throughputsequencing technology for high-throughput SNP detection is one of thecurrent research focuses.

Currently, second-generation sequencing technologies are needed to buildsequencing library. The sequencing libraries are then used forsequencing. The general steps include DNA extraction, DNA fragmentation,fragment selection, library construction (including adding connectors,amplification and other steps). The last step of machine sequencing isthe data analysis. Among these steps, library construction takes themost time and effort, and in the process of building a database, thesample genes are amplified multiple times and are therefore prone tobias. Building a database using this method, all the genome fragmentshave the same chance of being sequenced. Therefore, this method issuitable for genome sequencing. If only one of some genes or only aspecific part of the sequence is to be detected, this method would be awaste of sequencing space and would increase the difficulty of dataanalysis. In addition, samples treated in this way need complicatedsteps, need a huge complex sequencing data, require high initial amountof nucleic acid, and it is difficult for large-scale sequencing ofsamples simultaneously.

For some of the specific gene (such as exons, some single-genedisease-causing gene), sequencing will require additional steps tobuilding a database target sequence enrichment. Currently the mostwidely used method is to capture the enrichment of target sequences byhybridization. The widely used target sequence capture technology ismainly based on solid-phase hybridization (Choi et al. 2009) or liquidphase hybridization capture technology to capture (Bainbridge et al.2010). An existing custom capture commercial kit can be used (e.g.,NimbleGen sequence capture array or Aglient sureselect target enrichmentsystem, etc.), but these commercial custom sequence capture kits aregenerally expensive, and once the chips are customized, the targetsequences to be detected are fixed and cannot be changed. In addition tothe use of the target sequence capture technology for gene sequencingstudies, the PCR technique based on non-hybrid sequence capturetechnology has also been applied, but there's the disadvantage ofmultiplex PCR-based technologies. For example, some areas will not beeffectively amplified. Meanwhile, due to the amplification of errors bypolymerases, and that all of the genetic fragments are mixed andamplified, the sequencing results are difficult to verify.

Illumina offers a different PCR amplification method to build a database(TruSeq custom amplicon). Through the probe and target specific sequencehybridization, two probes are anchored to the target sequence at the 5′and 3′ ends. DNA polymerase extends to fill the gap (e.g., the sequenceof interest) between the two probes, followed by sequencing. This methodrequires design of different measured probes for the gene sequences. Itis complex and the quality of the database will be greatly influenced bythe hybridization efficiency. When using this method to detectlow-frequency mutations, it would waste sequencing space, because thevast majority of sequences being sequenced are wild-type sequences. Itneeds to increase the depth of sequencing to achieve compliance with therequirements of sensitivity. Using this method for large-scalepopulation mutated genetic screening, wild-type sequences will take upmost of the sequencing space, resulting in increased cost of sequencing.In addition, using this method, each sample requiring up to severalhundred ng amount of nucleic acid is not conducive to lowerconcentrations of some rare or difficult to obtain samples of nucleicacid sequencing.

In order to address the shortcomings of these approaches, there is aneed for a high-throughput sequencing method to build libraries for SNPdetection.

SUMMARY

The summary is not intended to be used to limit the scope of the claimedsubject matter. Other features, details, utilities, and advantages ofthe claimed subject matter will be apparent from the detaileddescription including those aspects disclosed in the accompanyingdrawings and in the appended claims.

In one aspect, disclosed herein is a probe set for analyzing a geneticlocus of a target polynucleotide sequence, comprising: one or more firstprobes comprising: (1) a first hybridization sequence that specificallybinds to the target polynucleotide sequence upstream of the geneticlocus, and (2) a first primer sequence upstream of the firsthybridization sequence, wherein the first primer sequence does not bindto the target polynucleotide sequence; and one or more second probescomprising: (i) a second hybridization sequence that specifically bindsto the target polynucleotide sequence downstream of the genetic locus,and (ii) a second primer sequence downstream of the second hybridizationsequence, wherein the second primer sequence does not bind to the targetpolynucleotide sequence, wherein: the extension directions of the firstand second probes are the same; and the first and second probes, whencoupled, form a sequence comprising the genetic locus.

In one embodiment, in a probe set herein, the genetic locus comprises anSNP or a point mutation; the first hybridization sequence of the one ormore first probes specifically binds to the target polynucleotidesequence upstream of the SNP or point mutation, and the 3′ terminalnucleotide of the one or more first probes is complementary to thenucleotide at the SNP or point mutation locus; and the secondhybridization sequence of the one or more second probes specificallybinds to the target polynucleotide sequence downstream of the SNP orpoint mutation, and the 5′ terminal nucleotide of the one or more secondprobes is complementary to the nucleotide immediately downstream of theSNP or point mutant locus.

In another embodiment, in a probe set herein, the genetic locuscomprises an insertion at the nth residue of a wild-type targetpolynucleotide sequence; the one or more first probes comprise at leasttwo first probes, one of which specifically binds to the wild-typetarget polynucleotide sequence until and excluding the nth residue,while the other specifically binds to the target polynucleotide sequenceincluding and until the last residue of the inserted sequence; and thesecond hybridization sequence of the one or more second probesspecifically binds to the target polynucleotide sequence downstream ofthe insertion, and the 5′ terminal nucleotide of the one or more secondprobes is complementary to nth residue.

In another embodiment, in a probe set herein, the genetic locuscomprises a deletion at the nth residue of a wild-type targetpolynucleotide sequence; the one or more first probes comprise at leasttwo first probes, one of which specifically binds to the wild-typetarget polynucleotide sequence until and excluding the nth residue,while the other specifically binds to the target polynucleotide sequenceuntil the first residue immediately upstream of the deleted sequence;and the second hybridization sequence of the one or more second probesspecifically binds to the target polynucleotide sequence downstream ofthe deletion, and the 5′ terminal nucleotide of the one or more secondprobes is complementary to nth residue.

In one aspect, provided herein is a probe set for analyzing a geneticlocus of a target polynucleotide sequence, comprising: one or more firstprobes comprising: (1) a first hybridization sequence that specificallybinds to the target polynucleotide sequence upstream of or including thegenetic locus, and (2) a first primer sequence upstream of the firsthybridization sequence, wherein the first primer sequence does not bindto the target polynucleotide sequence; and one or more second probescomprising: (i) a second hybridization sequence that specifically bindsto the target polynucleotide sequence downstream of or starting from thegenetic locus, and (ii) a second primer sequence downstream of thesecond hybridization sequence, wherein the second primer sequence doesnot bind to the target polynucleotide sequence, wherein the extensiondirections of the first and second probes are the same; the first probeis upstream of the second probe; and the first and second probes areadjacent and, when coupled, form a sequence comprising the geneticlocus.

In one embodiment, the genetic locus comprises an SNP or a pointmutation; and the first hybridization sequence of the one or more firstprobes specifically binds to the target polynucleotide sequenceincluding the SNP or point mutation, and the 3′ terminal nucleotide ofthe one or more first probes is complementary to the nucleotide at theSNP or point mutation locus, and the second hybridization sequence ofthe one or more second probes specifically binds to the targetpolynucleotide sequence downstream of the SNP or point mutation, and the5′ terminal nucleotide of the one or more second probes is complementaryto the nucleotide immediately downstream of the SNP or point mutantlocus; or the first hybridization sequence of the one or more firstprobes specifically binds to the target polynucleotide sequence upstreamof the SNP or point mutation, and the 3′ terminal nucleotide of the oneor more first probes is complementary to the nucleotide immediatelyupstream of the SNP or point mutation locus, and the secondhybridization sequence of the one or more second probes specificallybinds to the target polynucleotide sequence starting from the SNP orpoint mutation, and the 5′ terminal nucleotide of the one or more secondprobes is complementary to the nucleotide at the SNP or point mutationlocus.

In any of the preceding embodiments, the 5′ terminus of the one or moresecond probes can be phosphorylated. In any of the precedingembodiments, the first primer sequence of the one or more first probescan be a universal primer sequence.

In any of the preceding embodiments, the first primer sequence of theone or more first probes can uniquely identify the residue at the SNP ormutant locus.

In any of the preceding embodiments, the second primer sequence of theone or more second probes can be a universal primer sequence.

In one aspect, the genetic locus comprises a deletion or insertion.

In any of the preceding embodiments, the genetic locus can comprise aninsertion at the n^(th) residue of a wild-type target polynucleotidesequence; and the one or more first probes can comprise at least twofirst probes, one of which specifically binds to the wild-type targetpolynucleotide sequence until and excluding the n^(th) residue, whilethe other specifically binds to the target polynucleotide sequenceincluding and until the last residue of the inserted sequence, and thesecond hybridization sequence of the one or more second probesspecifically binds to the target polynucleotide sequence downstream ofthe insertion, and the 5′ terminal nucleotide of the one or more secondprobes is complementary to n^(th) residue; or the one or more secondprobes can comprise at least two second probes, one of whichspecifically binds to the wild-type target polynucleotide sequencestarting from the n^(th) residue, while the other specifically binds tothe target polynucleotide sequence including and from the first residueof the inserted sequence, and the first hybridization sequence of theone or more first probes specifically binds to the target polynucleotidesequence upstream of the insertion, and the 3′ terminal nucleotide ofthe one or more first probes is complementary to the residue immediatelyupstream of n^(th) residue in the wild-type target polynucleotide.

In any of the preceding embodiments, the genetic locus can comprise adeletion at the n^(th) residue of a wild-type target polynucleotidesequence; the one or more first probes can comprise at least two firstprobes, one of which specifically binds to the wild-type targetpolynucleotide sequence until and excluding the n^(th) residue, whilethe other specifically binds to the target polynucleotide sequence untilthe first residue immediately upstream of the deleted sequence, and thesecond hybridization sequence of the one or more second probesspecifically binds to the target polynucleotide sequence downstream ofthe deletion, and the 5′ terminal nucleotide of the one or more secondprobes is complementary to n^(th) residue; or the one or more secondprobes can comprise at least two second probes, one of whichspecifically binds to the wild-type target polynucleotide sequencestarting from the n^(th) residue, while the other specifically binds tothe target polynucleotide sequence including and from the first residueof the deleted sequence, and the first hybridization sequence of the oneor more first probes specifically binds to the target polynucleotidesequence upstream of the deletion, and the 5′ terminal nucleotide of theone or more first probes is complementary to the residue immediatelyupstream of n^(th) residue in the wild-type target polynucleotide.

In any of the preceding embodiments, the first primer sequence of theone or more first probes can be a universal primer sequence. In any ofthe preceding embodiments, the two first probes can comprise differentfirst primer sequences. In any of the preceding embodiments, the 5′terminus of the one or more second probes can be phosphorylated. In anyof the preceding embodiments, the second primer sequence of the one ormore second probes can be a universal primer sequence.

In any of the preceding embodiments, the genetic locus can be in adeafness related gene such as GJB2, SLC26A4, or 12SrRNA, wherein thegenetic locus optionally comprises 1494C>T, IVS7-2A>G, 235delC,176DEL16, and/or 299delAT. In one aspect, the one or more first probesfor 1494C>T comprises the polynucleotide sequence set forth in SEQ IDNO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and/or SEQ ID NO: 24. In any ofthe preceding embodiments, the one or more second probes for 1494C>T cancomprise the polynucleotide sequence set forth in SEQ ID NO: 25. In anyof the preceding embodiments, the one or more first probes for IVS7-2A>Gcan comprise the polynucleotide sequence set forth in SEQ ID NO: 7, SEQID NO: 8, SEQ ID NO: 9, and/or SEQ ID NO: 10. In any of the precedingembodiments, the one or more second probes for IVS7-2A>G can comprisethe polynucleotide sequence set forth in SEQ ID NO: 11. In any of thepreceding embodiments, the one or more first probes for 235delC cancomprise the polynucleotide sequence set forth in SEQ ID NO: 15 and/orSEQ ID NO:16. In any of the preceding embodiments, the one or moresecond probes for 235delC can comprise the polynucleotide sequence setforth in SEQ ID NO: 17. In any of the preceding embodiments, the one ormore first probes for 176DEL16 can comprise the polynucleotide sequenceset forth in SEQ ID NO: 12 and/or SEQ ID NO:13. In any of the precedingembodiments, the one or more second probes for 176DEL16 can comprise thepolynucleotide sequence set forth in SEQ ID NO: 14. In any of thepreceding embodiments, the one or more first probes for 299delAT cancomprise the polynucleotide sequence set forth in SEQ ID NO: 18 and/orSEQ ID NO:19. In any of the preceding embodiments, the one or moresecond probes for 299delAT can comprise the polynucleotide sequence setforth in SEQ ID NO: 20.

In one other aspect, provided herein is a kit for analyzing a geneticlocus, comprising the probe set of any of the preceding embodiments. Inone aspect, the kit further comprises a primer pair for amplifying176del16, 299delAT, and/or 235delC. In another aspect, the primer pairfor amplifying 176del16, 299delAT, and/or 235delC comprises thepolynucleotide sequences set forth in SEQ ID NO: 28 and SEQ ID NO:29. Inany of the preceding embodiments, the kit can further comprise a primerpair for amplifying 1494C>T. In one aspect, the primer pair foramplifying 1494C>T comprises the polynucleotide sequences set forth inSEQ ID NO: 26 and SEQ ID NO: 27. In any of the preceding embodiments,the kit can further comprise a primer pair for amplifying IVS7-2A>G. Inone aspect, the primer pair for amplifying IVS7-2A>G comprises thepolynucleotide sequences set forth in SEQ ID NO: 30 and SEQ ID NO: 31.In any of the preceding embodiments, one or both of the primers of theprimer pair can be labeled. In one aspect, the label comprises biotin.

In any of the preceding embodiments, the kit can further comprise abarcode specific primer and/or a common primer. In one aspect, thebarcode specific primer comprises the polynucleotide sequences set forthin SEQ ID NO: 32, and the common primer comprises the polynucleotidesequences set forth in SEQ ID NO: 33.

Also disclosed herein is a composition for analyzing at least a firstgenetic locus and a second genetic locus, comprising a first probe setof any one of any of the preceding embodiments for the first geneticlocus, and a second probe set of any of the preceding embodiments forthe second genetic locus. In one aspect, the probes in the first probeset are in equal molar amount, and the probes in the second probe setare in equal molar amount.

In any of the preceding embodiments, the first genetic locus cancomprise an SNP or point mutation, and the second genetic locus cancomprise a deletion and/or insertion. In one embodiment, the SNP orpoint mutation comprises 1494C>T, and/or IVS7-2A>G, and the deletionand/or insertion comprise 235delC, 176DEL16, and/or 299delAT.

In any of the preceding embodiments, the first and second probe sets cancomprise at least two of the following probe sets: (1) a probecomprising the polynucleotide sequences set forth in SEQ ID NO: 25, andone or more of a probe comprising the polynucleotide sequences set forthin SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24; (2) aprobe comprising the polynucleotide sequences set forth in SEQ ID NO:11, and one or more of a probe comprising the polynucleotide sequencesset forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10;(3) a probe comprising the polynucleotide sequences set forth in SEQ IDNO: 17, and one or more of a probe comprising the polynucleotidesequences set forth in SEQ ID NO: 15 or SEQ ID NO: 16; (4) a probecomprising the polynucleotide sequences set forth in SEQ ID NO: 14, andone or more of a probe comprising the polynucleotide sequences set forthin SEQ ID NO: 12 or SEQ ID NO: 13; and (5) a probe comprising thepolynucleotide sequences set forth in SEQ ID NO: 20, and one or more ofa probe comprising the polynucleotide sequences set forth in SEQ ID NO:18 or SEQ ID NO: 19.

In one aspect, provided herein is a method for analyzing a samplecomprising a target polynucleotide sequence comprising a genetic locus,comprising: (a) contacting the sample with a probe set, the probe setcomprising: one or more first probes comprising: (1) a firsthybridization sequence that specifically binds to the targetpolynucleotide sequence upstream of or including the genetic locus, and(2) a first primer sequence upstream of the first hybridizationsequence, wherein the first primer sequence does not bind to the targetpolynucleotide sequence; and one or more second probes comprising: (i) asecond hybridization sequence that specifically binds to the targetpolynucleotide sequence downstream of or starting from the geneticlocus, and (ii) a second primer sequence downstream of the secondhybridization sequence, wherein the second primer sequence does not bindto the target polynucleotide sequence, wherein the extension directionsof the first and second probes are the same, and the first probe isupstream of the second probe; (b) coupling the first and second probesbound to the target polynucleotide sequence to form a sequencecomprising the genetic locus; (c) determining the sequence comprisingthe genetic locus, thereby determining the sequence of the geneticlocus. In one aspect, the genetic locus comprises an SNP or a pointmutation; and the first hybridization sequence of the one or more firstprobes specifically binds to the target polynucleotide sequenceincluding the SNP or point mutation, and the 3′ terminal nucleotide ofthe one or more first probes is complementary to the nucleotide at theSNP or point mutation locus, and the second hybridization sequence ofthe one or more second probes specifically binds to the targetpolynucleotide sequence downstream of the SNP or point mutation, and the5′ terminal nucleotide of the one or more second probes is complementaryto the nucleotide immediately downstream of the SNP or point mutantlocus; or the first hybridization sequence of the one or more firstprobes specifically binds to the target polynucleotide sequence upstreamof the SNP or point mutation, and the 3′ terminal nucleotide of the oneor more first probes is complementary to the nucleotide immediatelyupstream of the SNP or point mutation locus, and the secondhybridization sequence of the one or more second probes specificallybinds to the target polynucleotide sequence starting from the SNP orpoint mutation, and the 5′ terminal nucleotide of the one or more secondprobes is complementary to the nucleotide at the SNP or point mutationlocus. In any of the preceding embodiments, the 5′ terminus of the oneor more second probes can be phosphorylated. In any of the precedingembodiments, the first primer sequence of the one or more first probescan be a universal primer sequence. In any of the preceding embodiments,the first primer sequence of the one or more first probes can be uniquefor the residue at the SNP or mutant locus. In any of the precedingembodiments, the second primer sequence of the one or more second probescan be a universal primer sequence.

In one aspect of a method disclosed herein, the genetic locus comprisesa deletion or insertion. In one aspect, the genetic locus comprises aninsertion at the n^(th) residue of a wild-type target polynucleotidesequence; and the one or more first probes comprise at least two firstprobes, one of which specifically binds to the wild-type targetpolynucleotide sequence until and excluding the n^(th) residue, whilethe other specifically binds to the target polynucleotide sequenceincluding and until the last residue of the inserted sequence, and thesecond hybridization sequence of the one or more second probesspecifically binds to the target polynucleotide sequence downstream ofthe insertion, and the 5′ terminal nucleotide of the one or more secondprobes is complementary to n^(th) residue; or the one or more secondprobes comprise at least two second probes, one of which specificallybinds to the wild-type target polynucleotide sequence starting from then^(th) residue, while the other specifically binds to the targetpolynucleotide sequence including and from the first residue of theinserted sequence, and the first hybridization sequence of the one ormore first probes specifically binds to the target polynucleotidesequence upstream of the insertion, and the 3′ terminal nucleotide ofthe one or more first probes is complementary to the residue immediatelyupstream of n^(th) residue in the wild-type target polynucleotide. Inanother aspect, the genetic locus comprises a deletion at the n^(th)residue of a wild-type target polynucleotide sequence; the one or morefirst probes comprise at least two first probes, one of whichspecifically binds to the wild-type target polynucleotide sequence untiland excluding the n^(th) residue, while the other specifically binds tothe target polynucleotide sequence until the first residue immediatelyupstream of the deleted sequence, and the second hybridization sequenceof the one or more second probes specifically binds to the targetpolynucleotide sequence downstream of the deletion, and the 5′ terminalnucleotide of the one or more second probes is complementary to n^(th)residue; or the one or more second probes comprise at least two secondprobes, one of which specifically binds to the wild-type targetpolynucleotide sequence starting from the n^(th) residue, while theother specifically binds to the target polynucleotide sequence includingand from the first residue of the deleted sequence, and the firsthybridization sequence of the one or more first probes specificallybinds to the target polynucleotide sequence upstream of the deletion,and the 5′ terminal nucleotide of the one or more first probes iscomplementary to the residue immediately upstream of n^(th) residue inthe wild-type target polynucleotide.

In any of the preceding embodiments, the first primer sequence of theone or more first probes can be a universal primer sequence. In any ofthe preceding embodiments, the two first probes can comprise differentfirst primer sequences. In any of the preceding embodiments, the 5′terminus of the one or more second probes can be phosphorylated. In anyof the preceding embodiments, the second primer sequence of the one ormore second probes can be a universal primer sequence. In any of thepreceding embodiments, the genetic locus can be in a deafness relatedgene such as GJB2, SLC26A4, or 12SrRNA, wherein the genetic locusoptionally comprises 1494C>T, IVS7-2A>G, 235delC, 176DEL16, and/or299delAT.

In any of the preceding embodiments, the one or more first probes for1494C>T can comprise the polynucleotide sequence set forth in SEQ ID NO:21, SEQ ID NO: 22, SEQ ID NO: 23, and/or SEQ ID NO: 24. In any of thepreceding embodiments, the one or more second probes for 1494C>T cancomprise the polynucleotide sequence set forth in SEQ ID NO: 25. In anyof the preceding embodiments, the one or more first probes for IVS7-2A>Gcan comprise the polynucleotide sequence set forth in SEQ ID NO: 7, SEQID NO: 8, SEQ ID NO: 9, and/or SEQ ID NO: 10. In any of the precedingembodiments, the one or more second probes for IVS7-2A>G can comprisethe polynucleotide sequence set forth in SEQ ID NO: 11. In any of thepreceding embodiments, the one or more first probes for 235delC cancomprise the polynucleotide sequence set forth in SEQ ID NO: 15 and/orSEQ ID NO:16. In any of the preceding embodiments, the one or moresecond probes for 235delC can comprise the polynucleotide sequence setforth in SEQ ID NO: 17. In any of the preceding embodiments, the one ormore first probes for 176DEL16 can comprise the polynucleotide sequenceset forth in SEQ ID NO: 12 and/or SEQ ID NO:13. In any of the precedingembodiments, the one or more second probes for 176DEL16 can comprise thepolynucleotide sequence set forth in SEQ ID NO: 14. In any of thepreceding embodiments, the one or more first probes for 299delAT cancomprise the polynucleotide sequence set forth in SEQ ID NO: 18 and/orSEQ ID NO:19. In any of the preceding embodiments, the one or moresecond probes for 299delAT can comprise the polynucleotide sequence setforth in SEQ ID NO: 20.

In any of the preceding embodiments, the method can further comprisepre-amplification of the target polynucleotide sequence before thecontacting step. In any of the preceding embodiments, thepre-amplification can comprise using a primer pair for amplifying adeafness related gene such as GJB2, SLC26A4, or 12SrRNA. In any of thepreceding embodiments, the genetic locus can comprise 1494C>T,IVS7-2A>G, 235delC, 176DEL16, and/or 299delAT.

In any of the preceding embodiments, the pre-amplification can compriseusing the primer pair having the polynucleotide sequences set forth inSEQ ID NO: 28 and SEQ ID NO: 29 to amplify 176del16, 299delAT, and/or235delC. In any of the preceding embodiments, the pre-amplification cancomprise using the primer pair having the polynucleotide sequences setforth in SEQ ID NO: 26 and SEQ ID NO: 27 to amplify 1494C>T. In any ofthe preceding embodiments, the pre-amplification can comprise using theprimer pair having the polynucleotide sequences set forth in SEQ ID NO:30 and SEQ ID NO: 31 to amplify IVS7-2A>G. In any of the precedingembodiments, one or both of the primers of the primer pair can belabeled. In any of the preceding embodiments, the label can comprisebiotin.

In any of the preceding embodiments, the coupling step can compriseligating the first and second probes bound to the target polynucleotidesequence. In any of the preceding embodiments, the determining step cancomprise amplification and/or sequencing of the coupled sequences, suchas high-throughput sequencing. In any of the preceding embodiments, theamplification and/or sequencing can comprise using a barcode specificprimer and/or a common primer. In any of the preceding embodiments, thebarcode specific primer can comprise the polynucleotide sequences setforth in SEQ ID NO: 32, and the common primer can comprise thepolynucleotide sequences set forth in SEQ ID NO: 33.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of the detection method according to oneaspect the present disclosure.

FIGS. 2A-2E are the testing results of plasmid group I. FIG. 2A is thetesting result of 1494 wild-type plasmid. FIG. 2B is the testing resultof IVS7-2 wild-type plasmid. FIG. 2C is the testing result of 176wild-type plasmid. FIG. 2D is the testing result of 235 wild typeplasmid. FIG. 2E is the testing result of 299 wild type plasmid.

FIGS. 3A-3E are the testing results of plasmid group II. FIG. 3A is thetesting result of position 1494 (C:T=1:1). FIG. 3B is the testing resultof position IVS7-2 (A:G=1:1). FIG. 3C is the testing result of position176 (WT:MT=3:1). FIG. 3D is the testing result of position 235(WT:MT=3:1). FIG. 3E is the testing result of position 299 (WT:MT=3:1).

FIGS. 4A-4E are the testing results of plasmid group III. FIG. 4A is thetesting result of 1494 mutant plasmid. FIG. 4B is the testing result ofIVS7-2 mutant plasmid. FIG. 4C is the testing result of position 176(WT:MT=2:1). FIG. 4D is the testing result of position 235 (WT:MT=2:1).FIG. 4E is the testing result of position 299 (WT:MT=2:1).

FIG. 5 is the testing result of 235 wild type genomic DNA and 235delChomozygous mutant genomic DNA.

DETAILED DESCRIPTION

A detailed description of one or more embodiments of the claimed subjectmatter is provided below along with accompanying figures that illustratethe principles of the claimed subject matter. The claimed subject matteris described in connection with such embodiments, but is not limited toany particular embodiment. It is to be understood that the claimedsubject matter may be embodied in various forms, and encompassesnumerous alternatives, modifications and equivalents. Therefore,specific details disclosed herein are not to be interpreted as limiting,but rather as a basis for the claims and as a representative basis forteaching one skilled in the art to employ the claimed subject matter invirtually any appropriately detailed system, structure, or manner.Numerous specific details are set forth in the following description inorder to provide a thorough understanding of the present disclosure.These details are provided for the purpose of example and the claimedsubject matter may be practiced according to the claims without some orall of these specific details. It is to be understood that otherembodiments can be used and structural changes can be made withoutdeparting from the scope of the claimed subject matter. It should beunderstood that the various features and functionality described in oneor more of the individual embodiments are not limited in theirapplicability to the particular embodiment with which they aredescribed. They instead can, be applied, alone or in some combination,to one or more of the other embodiments of the disclosure, whether ornot such embodiments are described, and whether or not such features arepresented as being a part of a described embodiment. For the purpose ofclarity, technical material that is known in the technical fieldsrelated to the claimed subject matter has not been described in detailso that the claimed subject matter is not unnecessarily obscured.

Unless defined otherwise, all terms of art, notations and othertechnical and scientific terms or terminology used herein are intendedto have the same meaning as is commonly understood by one of ordinaryskill in the art to which the claimed subject matter pertains. In somecases, terms with commonly understood meanings are defined herein forclarity and/or for ready reference, and the inclusion of suchdefinitions herein should not necessarily be construed to represent asubstantial difference over what is generally understood in the art.Many of the techniques and procedures described or referenced herein arewell understood and commonly employed using conventional methodology bythose skilled in the art.

All publications referred to in this application are incorporated byreference in their entireties for all purposes to the same extent as ifeach individual publication were individually incorporated by reference.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

Throughout this disclosure, various aspects of the claimed subjectmatter are presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theclaimed subject matter. Accordingly, the description of a range shouldbe considered to have specifically disclosed all the possible sub-rangesas well as individual numerical values within that range. For example,where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the claimed subject matter. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the claimed subjectmatter, subject to any specifically excluded limit in the stated range.Where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe claimed subject matter. This applies regardless of the breadth ofthe range. For example, description of a range such as from 1 to 6should be considered to have specifically disclosed sub-ranges such asfrom 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3to 6 etc., as well as individual numbers within that range, for example,1, 2, 3, 4, 5, and 6.

The practice of the provided embodiments will employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and sequencing technology,which are within the skill of those who practice in the art. Suchconventional techniques include polypeptide and protein synthesis andmodification, polynucleotide synthesis and modification, polymer arraysynthesis, hybridization and ligation of polynucleotides, and detectionof hybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the examples herein. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Green, et al., Eds., Genome Analysis: ALaboratory Manual Series (Vols. I-IV) (1999); Weiner, Gabriel, Stephens,Eds., Genetic Variation: A Laboratory Manual (2007); Dieffenbach,Dveksler, Eds., PCR Primer A Laboratory Manual (2003); Bowtell andSambrook, DNA Microarrays: A Molecular Cloning Manual (2003); Mount,Bioinformatics: Sequence and Genome Anazvsis (2004); Sambrook andRussell, Condensed Protocols from Molecular Cloning: A Laboratory Manual(2006); and Sambrook and Russell, Molecular Cloning: A Laboratory Manual(2002) (all from Cold Spring Harbor Laboratory Press); Ausubel et al.eds., Current Protocols in Molecular Biology (1987); T. Brown ed.,Essential Molecular Biology (1991), IRL Press; Goeddel ed., GeneExpression Technology (1991), Academic Press; A. Bothwell et al. eds.,Methods for Cloning and Analysis of Eukaryotic Genes (1990), BartlettPubl.; M. Kriegler, Gene Transfer and Expression (1990), Stockton Press;R. Wu et al. eds., Recombinant DNA Methodology (1989), Academic Press;M. McPherson et al., PCR: A Practical Approach (1991), IRL Press atOxford University Press; Stryer, Biochemistry (4th Ed.) (1995), W. H.Freeman, New York N.Y.; Gait, Oligonucleotide Synthesis: A PracticalApproach (2002), IRL Press, London; Nelson and Cox, Lehninger,Principles of Biochemistry (2000) 3rd Ed., W. H. Freeman Pub., New York,N.Y.; Berg, et al., Biochemistry (2002) 5th Ed., W. H. Freeman Pub., NewYork, N.Y., all of which are herein incorporated in their entireties byreference for all purposes.

As used herein, the singular forms “a”, “an”, and “the” include pluralreferences unless indicated otherwise. For example, “a” sample includesone or more samples.

It is understood that aspects and embodiments of the disclosuredescribed herein include “consisting” and/or “consisting essentially of”aspects and embodiments.

The term “binding” is used herein to refer to an attractive interactionbetween two molecules which results in a stable association in which themolecules are in close proximity to each other. Molecular binding can beclassified into the following types: non-covalent, reversible covalentand irreversible covalent. Molecules that can participate in molecularbinding include polypeptides, polynucleotides, carbohydrates, lipids,and small organic molecules such as pharmaceutical compounds.Polypeptides that form stable complexes with other molecules are oftenreferred to as receptors while their binding partners are calledligands. Polynucleotides can also form stable complex with themselves orothers, for example, DNA-protein complex, DNA-DNA complex, DNA-RNAcomplex.

The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and“nucleic acid molecule” are used interchangeably herein to refer to apolymeric form of nucleotides of any length, and may compriseribonucleotides, deoxyribonucleotides, analogs thereof, or mixturesthereof. This term refers only to the primary structure of the molecule.Thus, the term includes triple-, double- and single-strandeddeoxyribonucleic acid (“DNA”), as well as triple-, double- andsingle-stranded ribonucleic acid (“RNA”). It also includes modified, forexample by alkylation, and/or by capping, and unmodified forms of thepolynucleotide. More particularly, the terms “polynucleotide,”“oligonucleotide,” “nucleic acid” and “nucleic acid molecule” includepolydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA,and mRNA, whether spliced or unspliced, any other type of polynucleotidewhich is an N- or C-glycoside of a purine or pyrimidine base, and otherpolymers containing normucleotidic backbones, for example, polyamide(e.g., peptide nucleic acid (“PNA”)) and polymorpholino (commerciallyavailable from the Anti-Virals, Inc., Corvallis, Oreg., as Neugene)polymers, and other synthetic sequence-specific nucleic acid polymersproviding that the polymers contain nucleobases in a configuration whichallows for base pairing and base stacking, such as is found in DNA andRNA. Thus, these terms include, for example, 3′-deoxy-2′,5′-DNA,oligodeoxyribonucleotide N3′ to P5′ phosphoramidates,2′-O-alkyl-substituted RNA, hybrids between DNA and RNA or between PNAsand DNA or RNA, and also include known types of modifications, forexample, labels, alkylation, “caps,” substitution of one or more of thenucleotides with an analog, intemucleotide modifications such as, forexample, those with uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoramidates, carbamates, etc.), with negativelycharged linkages (e.g., phosphorothioates, phosphorodithioates, etc.),and with positively charged linkages (e.g., aminoalkylphosphoramidates,aminoalkylphosphotriesters), those containing pendant moieties, such as,for example, proteins (including enzymes (e.g. nucleases), toxins,antibodies, signal peptides, poly-L-lysine, etc.), those withintercalators (e.g., acridine, psoralen, etc.), those containingchelates (of, e.g., metals, radioactive metals, boron, oxidative metals,etc.), those containing alkylators, those with modified linkages (e.g.,alpha anomeric nucleic acids, etc.), as well as unmodified forms of thepolynucleotide or oligonucleotide.

It will be appreciated that, as used herein, the terms “nucleoside” and“nucleotide” will include those moieties which contain not only theknown purine and pyrimidine bases, but also other heterocyclic baseswhich have been modified. Such modifications include methylated purinesor pyrimidines, acylated purines or pyrimidines, or other heterocycles.Modified nucleosides or nucleotides can also include modifications onthe sugar moiety, e.g., wherein one or more of the hydroxyl groups arereplaced with halogen, aliphatic groups, or are functionalized asethers, amines, or the like. The term “nucleotidic unit” is intended toencompass nucleosides and nucleotides.

“Nucleic acid probe” and “probe” are used interchangeably and refer to astructure comprising a polynucleotide, as defined above, that contains anucleic acid sequence that can bind to a corresponding target. Thepolynucleotide regions of probes may be composed of DNA, and/or RNA,and/or synthetic nucleotide analogs.

As used herein, “complementary or matched” means that two nucleic acidsequences have at least 50% sequence identity. Preferably, the twonucleic acid sequences have at least 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% of sequence identity. “Complementary or matched” alsomeans that two nucleic acid sequences can hybridize under low, middleand/or high stringency condition(s). The percentage of sequence identityor homology is calculated by comparing one to another when aligned tocorresponding portions of the reference sequence.

As used herein, “substantially complementary or substantially matched”means that two nucleic acid sequences have at least 90% sequenceidentity. Preferably, the two nucleic acid sequences have at least 95%,96%, 97%, 98%, 99% or 100% of sequence identity. Alternatively,“substantially complementary or substantially matched” means that twonucleic acid sequences can hybridize under high stringency condition(s).The percentage of sequence identity or homology is calculated bycomparing one to another when aligned to corresponding portions of thereference sequence.

In general, the stability of a hybrid is a function of the ionconcentration and temperature. Typically, a hybridization reaction isperformed under conditions of lower stringency, followed by washes ofvarying, but higher, stringency. Moderately stringent hybridizationrefers to conditions that permit a nucleic acid molecule such as a probeto bind a complementary nucleic acid molecule. The hybridized nucleicacid molecules generally have at least 60% identity, including forexample at least any of 70%, 75%, 80%, 85%, 90%, or 95% identity.Moderately stringent conditions are conditions equivalent tohybridization in 50% formamide, 5×Denhardt's solution, 5×SSPE, 0.2% SDSat 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 42° C. Highstringency conditions can be provided, for example, by hybridization in50% formamide, 5×Denhardt's solution, 5×SSPE, 0.2% SDS at 42° C.,followed by washing in 0.1×SSPE, and 0.1% SDS at 65° C. Low stringencyhybridization refers to conditions equivalent to hybridization in 10%formamide, 5×Denhardt's solution, 6×SSPE, 0.2% SDS at 22° C., followedby washing in 1×SSPE, 0.2% SDS, at 37° C. Denhardt's solution contains1% Ficoll, 1% polyvinylpyrolidone, and 1% bovine serum albumin (BSA).20×SSPE (sodium chloride, sodium phosphate, ethylene diamide tetraaceticacid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and0.025 M EDTA. Other suitable moderate stringency and high stringencyhybridization buffers and conditions are well known to those of skill inthe art and are described, for example, in Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press,Plainview, N.Y. (1989); and Ausubel et al., Short Protocols in MolecularBiology, 4th ed., John Wiley & Sons (1999).

Alternatively, substantial complementarity exists when an RNA or DNAstrand will hybridize under selective hybridization conditions to itscomplement. Typically, selective hybridization will occur when there isat least about 65% complementary over a stretch of at least 14 to 25nucleotides, preferably at least about 75%, more preferably at leastabout 90% complementary. See M. Kanehisa Nucleic Acids Res. 12:203(1984).

The terms “homologous”, “substantially homologous”, and “substantialhomology” as used herein denote a sequence of amino acids having atleast 50%, 60%, 70%, 80% or 90% identity wherein one sequence iscompared to a reference sequence of amino acids. The percentage ofsequence identity or homology is calculated by comparing one to anotherwhen aligned to corresponding portions of the reference sequence.

In one aspect, provided herein is a method based on sequencing, such ashigh-throughput sequencing, to detect an SNP or mutation in a sample. Insome embodiments, the mutation can include a point mutation, a deletion,an insertion, an indel, or a combination thereof.

In one embodiment, the method comprises designing one or more probesand/or one or more pre-amplification primer pairs, to detect one or moretarget sequences comprising the SNP(s) and/or mutation site(s). In anyof the preceding embodiments, the mutation site(s) can be an SNP, apoint mutation, an insertion, and/or a deletion.

In some embodiments, the one or more probes comprise one or moredetection probes. In any of the preceding embodiments, the one or moredetection probes can include one or more detection probes A fordetecting the SNP(s) or point mutation(s). In any of the precedingembodiments, the one or more detection probes can further include one ormore detection probes B for detecting the insertion(s) and/ordeletion(s).

In any of the preceding embodiments, each of the detection probes A cancorrespond to and specifically detect an SNP or point mutation. Forexample, detection probes A for position IVS7-2 in the SLC26A1 gene anddetection probes A for position 1494 of the 12SrRNA gene can be used inone or more reaction runs to detect the corresponding SNP or pointmutation in a sample. The detection probes A for a specific SNP or pointmutation position can comprise one or more probes ending in A, T, C, orG.

In any of the preceding embodiments, each of the detection probes B cancorrespond to and specifically detect an insertion or deletion.

In any of the preceding embodiments, the detection probes A fordetecting one or more SNP sites can comprise one or more upstreamgenotyping probes ASO-A and one or more downstream site-specific probesLSO-A. In some embodiments, the ASO-A probe(s) and the LSO-A probe(s)have the same extending direction. In any of the preceding embodiments,the 3′end of an ASO-A probe can be identical or complementary to thetarget sequence of the SNP site (either wild-type or mutant). Forexample, two ASO-A probes can be used, one having a 3′end that isidentical or complementary to the wild-type target sequence of the SNPsite, and the other having a 3′end that is identical or complementary tothe mutant target sequence of the SNP site.

In any of the preceding embodiments, the 3′ terminal nucleotide of anASO-A probe can be identical or complementary to the nucleotide at thewild-type or mutant SNP site. In any of the preceding embodiments, the5′ end of the ASO-A probe can be free from the target sequence of theSNP site. In another aspect, the 5′ end of the ASO-A probe is separatedfrom the target sequence. In some aspects, the ASO-A probe comprises a5′ end sequence that is not identical or complimentary to the targetsequence, and the 5′ end sequence does not hybridize to the targetsequence or a complementary sequence thereof. In some aspects, the 5′end sequence comprises a common probe sequence P1.

In any of the preceding embodiments, each SNP or mutation can correspondto an ASO-A probe. For example, for a point mutation site where all fourof A, T, C, or G is possible, four ASO-A probes can be designed foranalyzing the site in a sample. Any one or more of the four ASO-A probescan be used in a reaction. Thus, the base at 3′ end of the four ASO-Aprobes can be A, T, C, or G, respectively. The 5′ end of each of theASO-A probes is free from the target sequence and comprises a universalprobe P1.

In some embodiments, three of the four ASO-A probes can be used. Forexample, the bases at the 3′ ends of two ASO-A probes are complementaryto the SNP site in the wild-type or mutant status, respectively. Thethird ASO-A probe comprises a 3′ terminal nucleotide that is differentfrom the 3′ terminal nucleotides of the first two ASO-A probes. The 5′end of each of the ASO-A probes is free from the target sequence andcomprises a universal probe P1.

In some embodiments, two of the four ASO-A probes can be used. Forexample, the bases at the 3′ ends of the two ASO-A probes arecomplementary to the SNP site in the wild-type or mutant status,respectively. The 5′ end of each of the ASO-A probes is free from thetarget sequence and comprises a universal probe P1.

In some embodiments, one of the four ASO-A probes is used. The 3′terminal nucleotide of the ASO-B A probe is identical or complementaryto the nucleotide at the SNP or mutation to be detected.

In any of the preceding embodiments, the 5′ end of the LSO-A probe canbe identical or complementary to the target sequence. In one aspect, the5′ terminal nucleotide of the LSO-A probe is identical or complementaryto the first nucleotide of the target sequence immediately downstream ofthe SNP site in the direction of probe extension.

In any of the preceding embodiments, the 3′end of the LSO-A probe can befree from the target sequence of the SNP site. In another aspect, the 3′end of the LSO-A probe is separated from the target sequence. In someaspects, the LSO-A probe comprises a 3′ end sequence that is notidentical or complimentary to the target sequence, and the 3′ endsequence does not hybridize to the target sequence or a complementarysequence thereof. In some aspects, the 3′ end sequence comprises acommon probe sequence P2.

In any of the preceding embodiments, the 5′ end of the LSO-A probe canbe phosphorylated. In one aspect, the 5′ terminal nucleotide of theLSO-A probe is phosphorylated.

In any of the preceding embodiments, the detection probes B fordetecting the insertion/deletion mutation site can comprise an upstreamgenotyping probe ASO-B for detecting the insertion/deletion mutation, anupstream genotyping probe ASO-B for detecting the wild-type sequence,and a downstream site-specific probe LSO-B. The three probes can havethe same extending direction.

In one aspect, the upstream genotyping probe ASO-B for detecting theinsertion/deletion mutation comprises an upstream genotyping probe ASO-Bfor detecting an insertion, and/or an upstream genotyping probe ASO-Bfor detecting a deletion.

In any of the preceding embodiments, the 3′ end of the upstreamgenotyping probe ASO-B probe for detecting an insertion can comprise asequence that is identical or complementary to the target sequence,and/or a sequence that is identical or complementary to the insertionsequence. In one aspect, the 3′ terminal nucleotide of the ASO-B probeis identical or complementary to the last nucleotide of the insertedsequence, for example, the 5′ terminal nucleotide of the insertedsequence.

The 3′ end of the upstream genotyping probe ASO-B probe for detectingthe wild-type sequence can be identical or complementary to thewild-type target sequence without the insertion, and in one aspect, the3′ terminal nucleotide of the ASO-B probe is identical or complementaryto the nucleotide in the wild-type sequence immediately upstream of theinsertion site.

In any of the preceding embodiments, the 3′ end of the upstreamgenotyping probe ASO-B probe for detecting a deletion can comprise asequence that is identical or complementary to the target sequenceupstream of the deleted sequence, and/or a sequence that is identical orcomplementary to the target sequence downstream of the deleted sequence.In one aspect, the 3′ terminal nucleotide of the ASO-B probe isidentical or complementary to the nucleotide in the wild-type sequencethat is immediately 5′ to the first nucleotide of the deleted sequence.In one aspect, the 3′ terminal nucleotide of the ASO-B probe isidentical or complementary to the nucleotide in the wild-type sequencethat is immediately upstream of the first nucleotide (in the 5′ to 3′direction) of the deleted sequence. In one aspect, the 3′ terminalnucleotide of the ASO-B probe is identical or complementary to the firstupstream nucleotide next to the deletion site.

In some aspects, the target sequence of the insertion/deletion mutationsite is the wild-type target sequence with insertion or deletion beforethe n^(th) base.

In any of the preceding embodiments, the 5′ end of the downstreamsite-specific probe LSO-B can be identical or complementary to thetarget sequence of the wild-type site, and the 5′ end nucleotide can beidentical or complementary to the n^(th) nucleotide of the wild-typesite sequence in the probe extending direction.

In any of the preceding embodiments, the 3′ end of the LSO-B probe canbe free from the target sequence and comprise a universal probe P2.

In any of the preceding embodiments, each upstream genotyping probe ASOand the corresponding downstream site-specific probe LSO of an SNP ormutation site can be coupled together without overlap or gap to obtain asequence comprising the genetic locus to be analyzed. In one aspect, thesequence comprises a reverse complement sequence of the target sequence.

In any of the preceding embodiments, the coupling of the first andsecond probes can be performed by ligation. In other embodiments, thecoupling can be performed by using one or more adaptors (such as anadaptor sequence that hybridizes to the target sequence in between thefirst and second probes), and then ligating the first probe to theadaptor and the adaptor to the second probe.

In any of the preceding embodiments, the pre-amplification primer paircan comprise one or more pre-amplification primer pairs. In one aspect,the pre-amplification primer pairs are capable of amplifying targetsequences comprising one or more target SNP or mutation sites.

In any of the preceding embodiments, the 5′ end of one primer in each ofthe pre-amplification primer pairs can be labeled, for example, withbiotin.

In any of the preceding embodiments, the method disclosed herein canfurther comprise a step of pre-amplifying the test sample(s) using oneor more of the pre-amplification primer pairs disclosed herein, forexample, to obtain biotin-labeled polynucleotides of the targetsequences.

In any of the preceding embodiments, the method disclosed herein canfurther comprise a step of using one or more of the detection probes A,a mixture of the detection probes A, one or more of the detection probesB, and/or a mixture of the detection probes B, to hybridize with thepre-amplified polynucleotides of the target sequences (such as thebiotin-labeled nucleic acids) and obtain hybridization products.

In any of the preceding embodiments, the method disclosed herein canfurther comprise a step of ligating the hybridization products using aDNA ligase, to obtain one or more sequencing target sequences.

In any of the preceding embodiments, the method disclosed herein canfurther comprise a step of amplifying and sequencing the target sequenceusing a barcode specific primer and a common primer to obtain sequencingresult of the target amplification products. In one aspect, the 20 basesof the 3′ end of the barcode primer and the common primer arecomplementary to the universal probe sequence P1 or P2, respectively. Inanother aspect, the barcode specific primer comprises a barcode sequenceXXXXXX, which is a random hexamer sequence of A, T, C, and G, and eachcombination can be used to identify a different sample.

In any of the preceding embodiments, the method disclosed herein canfurther comprise a step of analyzing the sequencing results to determinewhether the test sample contains the mutation site and/or to determinethe genotype(s) of the mutation site(s).

The analysis of the sequencing results to determine whether the testsample containing the mutation site or to determine the mutation sitegenotype can comprise comparing the reverse complementary sequence of atarget sequence comprising a mutation with the sequencing results. Thenumber of the sequences in the sequencing results having the samesequence as the reverse complementary sequence of the target sequence isrecorded as the copy number of the mutation. The ratio of the mutationcopy number in the total number of sequencing results is calculated. Thegenotype information of the site in the sample can then be determinedbased on the ratio.

In one aspect, analyzing the genotype information of the site is basedon known information and statistical analysis, by setting an appropriatethreshold, and determining the locus information according to thethreshold.

In one embodiment, if the mutation site ratio is greater than 90%, thetest sample contains or likely contains the mutation site, or thegenotype of test sample is homozygous of the mutation site or acandidate of homozygous mutation site. If the mutation site ratio is20-90%, the test sample contains or likely contains the mutation site,or the genotype of test sample is a heterozygous mutation site or acandidate of heterozygous mutation site. If the mutation site ratio isless than 20%, the test sample does not contain or likely does notcontain the mutation site, or the genotype of test sample is wild-typeor a candidate for wild-type sample.

The analysis of the sequencing results can comprise comparing thereverse complementary sequence of a wild-type target sequence with thesequencing results. The number of the sequences in the sequencingresults having the same sequence as the reverse complementary sequenceof the wild-type target sequence is recorded as the copy number of thewild-type sequences. The ratio of the wild-type copy number in the totalnumber of sequencing results is calculated. The genotype information ofthe site in the sample can then be determined based on the ratio. Thetotal number of sequencing results is equal to the total of thewild-type copy number and the mutant copy number.

In any of the preceding embodiments, the mutation site can be a SNP siteand/or insertion/deletion mutation site.

In any of the preceding embodiments, the detection probe can comprise aplurality of detection probes A (for SNP or point mutation loci) and/ora plurality of detection probes B (for deletion and/or insertion).

In any of the preceding embodiments, each probe can be mixed inequimolar ratio.

In any of the preceding embodiments, the pre-amplification primers cancomprise an equimolar mixture of the plurality of pre-amplificationprimer pairs.

In any of the preceding embodiments, the sample can be one or moresamples, and each barcode specific primer can correspond to one sample.

In any of the preceding embodiments, the detection probes A can be amixture of a plurality of the detection probes A in equimolar ratio.

In any of the preceding embodiments, the detection probes B can be amixture of a plurality of the detection probes B in equimolar ratio.

In any of the preceding embodiments, after pre-amplification, the methodcan further comprise adding exonuclease I and/or alkaline phosphatase tothe pre-amplification product to remove a pre-amplification primer anddNTP in the system, for example, to obtain the biotin-labeled nucleicacids.

In any of the preceding embodiments, the method can further compriseusing magnetic beads to adsorb and capture the hybridization products.

In any of the preceding embodiments, the sequencing step can compriseusing a second-generation sequencing method.

In any of the preceding embodiments, the detection probes can comprisetwo detection probes A for detecting an SNP or mutation, and threedetection probes B for detecting a deletion and/or insertion.

In any of the preceding embodiments, the SNP sites can be 1494C>T and/orIVS7-2A>G.

In any of the preceding embodiments, the deletion or insertion mutationsite can be 235delC, 176DEL16, and/or 299delAT.

In any of the preceding embodiments, detection probes A can compriseprobes for 1494C>T. In one aspect, the probes can comprise an ASO-Aprobe comprising the polynucleotide sequence shown in SEQ ID NO: 21, anASO-A probe comprising the polynucleotide sequence shown in SEQ ID NO:22, an ASO-A probe comprising the polynucleotide sequence shown in SEQID NO: 23, and/or an ASO-A probe comprising the polynucleotide sequenceshown in SEQ ID NO: 24. In any of the preceding embodiments, the probescan comprise a LSO-A probe, for example, a probe comprising thepolynucleotide sequence shown in SEQ ID NO: 25.

In any of the preceding embodiments, detection probes A can compriseprobes for IVS7-2A>G. In one aspect, the probes can comprise an ASO-Aprobe comprising the polynucleotide sequence shown in SEQ ID NO: 7, anASO-A probe comprising the polynucleotide sequence shown in SEQ ID NO:8, an ASO-A probe comprising the polynucleotide sequence shown in SEQ IDNO: 9, and/or an ASO-A probe comprising the polynucleotide sequenceshown in SEQ ID NO: 10. In any of the preceding embodiments, the probescan comprise a LSO-A probe, for example, a probe comprising thepolynucleotide sequence shown in SEQ ID NO: 11.

In any of the preceding embodiments, detection probes B can compriseprobes for 235delC. In one aspect, the probes can comprise an ASO-Bprobe comprising the polynucleotide sequence shown in SEQ ID NO: 15,and/or an ASO-B probe comprising the polynucleotide sequence shown inSEQ ID NO: 16. In any of the preceding embodiments, the probes cancomprise a LSO-B probe, for example, a probe comprising thepolynucleotide sequence shown in SEQ ID NO: 17.

In any of the preceding embodiments, detection probes B can compriseprobes for 176DEL16. In one aspect, the probes can comprise an ASO-Bprobe comprising the polynucleotide sequence shown in SEQ ID NO: 12,and/or an ASO-B probe comprising the polynucleotide sequence shown inSEQ ID NO: 13. In any of the preceding embodiments, the probes cancomprise a LSO-B probe, for example, a probe comprising thepolynucleotide sequence shown in SEQ ID NO: 14.

In any of the preceding embodiments, detection probes B can compriseprobes for 299delAT. In one aspect, the probes can comprise an ASO-Bprobe comprising the polynucleotide sequence shown in SEQ ID NO: 18,and/or an ASO-B probe comprising the polynucleotide sequence shown inSEQ ID NO: 19. In any of the preceding embodiments, the probes cancomprise a LSO-B probe, for example, a probe comprising thepolynucleotide sequence shown in SEQ ID NO: 20.

In any of the preceding embodiments, the pre-amplification primers cancomprise a primer pair for amplifying 176del16, 299delAT, and/or235delC.

In any of the preceding embodiments, the pre-amplification primers cancomprise a primer pair for amplifying 1494C>T.

In any of the preceding embodiments, the pre-amplification primers cancomprise a primer pair for amplifying IVS7-2A>G.

In any of the preceding embodiments, the pre-amplification primers cancomprise a primer pair having one primer comprising the single strandpolynucleotide sequence of SEQ ID NO: 28 and another primer comprisingthe single strand polynucleotide sequence of SEQ ID NO: 29.

In any of the preceding embodiments, the pre-amplification primers cancomprise a primer pair having one primer comprising the single strandpolynucleotide sequence of SEQ ID NO: 26 and another primer comprisingthe single strand polynucleotide sequence of SEQ ID NO: 27.

In any of the preceding embodiments, the pre-amplification primers cancomprise a primer pair having one primer comprising the single strandpolynucleotide sequence of SEQ ID NO: 30 and another primer comprisingthe single strand polynucleotide sequence of SEQ ID NO: 31.

In one aspect, disclosed herein is a kit for detecting one or more SNPsor mutation sites. In one embodiment, the kit comprises one or moredetection probes A for detecting 1494C>T, one or more detection probes Afor detecting IVS7-2A>G, one or more detection probes B for detecting235delC, one or more detection probes B for detecting 176DEL16, and/orone or more detection probes B for detecting 299delAT. In any of thepreceding embodiments, the kit can further comprise a pre-amplificationprimer pair for amplifying 176del16, 299delAT, and/or 235delC, apre-amplification primer pair for amplifying 1494C>T, apre-amplification primer pair for amplifying IVS7-2A>G.

In any of the preceding embodiments, the mutation sites can comprise1494C>T, IVS7-2A>G, 235delC, 176DEL16, and/or 299delAT.

In any of the preceding embodiments, the barcode primer nucleotidesequence can comprise the polynucleotide sequence of SEQ ID NO: 32.

In any of the preceding embodiments, the common primer nucleotidesequence can comprise the polynucleotide sequence of SEQ ID NO: 33.

In any of the preceding embodiments, detecting a plurality of SNPsand/or mutations can performed using a method disclosed herein. Forexample, a plurality of pre-amplification primers can be mixed toamplify different target polynucleotides in a sample. In another aspect,a plurality of detection probes can be mixed and hybridized to differenttarget polynucleotides in the sample.

In any of the preceding embodiments, the mutation sites can comprise235delC (deletion of C), 176del16 (deletion of 16 bp), 299delAT(deletion of AT), IVS7-2A>G (A mutated to G), and/or 1494C>T (C mutatedto T).

In any of the preceding embodiments, the method can further comprisehybridization and capture of hybridization products by beads. In oneaspect, appropriate concentrations of the detection probes ASO and LSOand a sample (with to without pre-treatment) are added to the reactionsystem. In another aspect, hybridization solution or binding buffer withappropriate salt concentrations are added. Then the reaction is subjectto denaturation under high temperature, and then annealing underlow-temperature, so that the ASO and LSO probe sequences and thesequences in the target polynucleotides around the SNP or mutation siteare allowed to fully hybridize. After annealing is completed, beads areadded, and the reaction is subjected to incubation under a suitabletemperature. After completion of the incubation, a washing buffer andTaq ligation buffer are used to wash the beads to remove unhybridizedprobes.

After the washing is complete, a DNA ligase is used to ligate the probesbound to the target sequences at a suitable temperature. In one aspect,after coupling of the probes after ligation is completed, the ligationsolution is discarded, and the beads are re-suspended in a suitableamount of ddH2O. Then, the re-suspended beads are heated to dissociatethe ligated probes.

In one aspect, the ligated probes are amplified and/or sequenced usingthe barcode primer and/or the common primer to amplify, for example, byPCR.

In one aspect, the sample can be divided into two portions forhybridization. For one of the portions, only detection probes for thewild-type sequence are added for hybridization. For the other portion,detection probes for the mutant sequence (for example, for an SNP orpoint mutation, the other three probes except the wild-type probe) areadded for hybridization. The samples are then subjected to ligation andamplified using the barcode primer and/or the common primer. Theamplification products can be quantified, and the portions can be mixedaccording to a known ratio, which mixture is then subjected tosequencing, for example, high-throughput sequencing. This way, when suchlarge-scale SNP mutation screening is used for detection of lowfrequency mutations, mutation-specific probes can be used only to detectmutant loci, thereby effectively using sequencing space and saving thecost of sequencing.

In some aspects, the technical improvements provided by a method hereininclude the following:

(1) The introduction of the pre-amplification step greatly increases thesensitivity of the system, such that nucleic acids lower than 50 copiescould be detected.

(2) The sample labeling step is simple. The sample can be labeled duringthe progress of pre-amplification, without requiring additional labelingstep.

(3) After pre-amplification, specific probes for detecting are used andenriched. Compared to the amplicon sequencing method, the present methodexcludes the impact of non-specific amplification product inpre-amplification. This is helpful for data analysis.

(4) The method of building a database is simple. Genomic fragments andartificial joints and other tedious steps are not required.

(5) Using a DNA ligase directly distinguishes SNP nucleotide and avoidsthe process of random mutation caused by extension, thereby improvingthe accuracy of sequencing.

(6) The probes can be used for large-scale sample screening, greatlyreducing the cost of sequencing.

(7) Since all sequences are known, data analysis is easy.

(8) Site selection is flexible. The method is ideal for a moderatenumber of mutations for large-scale sample screening.

In one aspect, based on the feature of DNA ligases, the presentdisclosure provides a method of library construction, designing twoadjacent probes according to the SNP site information. In one aspect,the 3′ end of one of the probes corresponds to the SNP site. In oneaspect, when two adjacent probes are completely complementary to the SNPsite sequence, they can be ligated by a DNA ligase; otherwise it willnot be ligated. By using the universal primer sequences in the probes,PCR amplification and library construction can be conducted. Then asecond-generation sequencing technology can be used for high-throughputsequencing and analysis of the SNP site sequence information. In oneaspect, simultaneous analysis of multiple loci can be achieved through acombination of different probes to detect multiple sites. Using barcodeprimers, samples can be mixed and each sample is identified by a barcodeprimer. For example, samples can be mixed according to known ratios andthen sequenced at the same time, achieving multi-site, multi-sample andhigh-throughput testing. When carrying out large-scale populationscreening for mutations in the gene, mutant probe may be used alone todetect with more efficient use of sequencing space and lower cost.

Various embodiments in the device of the present disclosure aredescribed in a progressive manner. Differences between variousembodiments are emphasized in their specifications, while their commonstructures can be referred from the description.

Embodiment 1: A method based on high-throughput sequencing to detect themutation site in test sample, comprising:

1) design probes and pre-amplification primer pairs to detect targetsequences of known mutation sites, Said mutation sites are the SNP sitesand insertions/deletions; Said detection probes are one or moredetection probes A for detecting the SNP loci and/or one or moredetection probes B for detecting insertions or deletion; said each probeA or probe B is corresponding to one of the mutation sites; Saiddetection probes A for detecting SNP sites comprise upstream genotypingprobes ASO-A and downstream site-specific probes LSO-A, and said ASO-Aprobes and said LSO-A probes have the same extending direction; 3′ endof said ASO-A probe is identical or complementary to the target sequenceof the SNP site in wild-type or mutant; 3′ terminal nucleotide of saidASO-A probe is identical or complementary to the SNP site in wild-typeor mutant; 5′ end of said ASO-A probe is free from the target sequenceof the SNP site in wild-type or mutant, and a universal probe P1; Eachsaid SNP mutations is corresponding to an ASO-A probe; 5′ end of saidLSO-A probe is identical or complementary to the target sequence of theSNP site in wild-type or mutant; 5′ terminal nucleotide of said LSO-Aprobe, with the terminal nucleotide phosphorylation, is identical orcomplementary to the first nucleotide of target sequence of SNP site inthe direction of probe extension, 3′ end of said LSO-A probe is freefrom the target sequence, and a universal probe P2; Said detectionprobes B for detecting insertion/deletion mutation sites comprise aupstream genotyping probe ASO-B for detecting insertion/deletionmutations, a upstream genotyping probe ASO-B for detecting wild-type,and a downstream site-specific probe LSO-B; said three probes have thesame extending direction; Said upstream genotyping probe ASO-B fordetecting insertion/deletion mutations is upstream genotyping probesASO-B for detecting insertion or upstream genotyping probes ASO-B fordetecting deletion. 3′ end of said upstream genotyping probe ASO-B probefor detecting insertion is identical or complementary to the targetsequence of insertion site in wild-type or mutant; 3′ terminalnucleotide of said ASO-B probe is identical or complementary to the lastnucleotide of the insertion site; 3′ end of said upstream genotypingprobe ASO-B probe for detecting deletion is identical or complementaryto the target sequence of deletion site in wild-type or mutant; 3′terminal nucleotide of said ASO-B probe is identical or complementary tothe first upstream nucleotide next to the deletion site; Target sequenceof insertion/deletion mutation sites is the wild-type target sequencewith insertion or deletion before the nth bases. 5′ end of thedownstream site-specific probe LSO-B is identical or complementary tothe target sequence of the wild-type site, and 5′ end nucleotide isidentical or complementary to the nth nucleotide of the wild-type sitesequence in probe extending direction; 3′ end of said LSO-B probe isfree from the target sequence, and a universal probe P2; Each upstreamgenotyping probe ASO and corresponding downstream site-specific probeLSO of a mutation site splice together without overlapping or gap,obtaining the reverse complement sequence of the target sequence; Saidpre-amplification primer pairs are one or more pre-amplification primerpair, said pre-amplification primer pairs are capable of amplifying theone or more target mutation sites; and 5′ end of each of saidpre-amplification primer pair is labeled with biotin;

2) conduct pre-amplification of the test samples by one or morepre-primer pairs to obtain a biotin labeled nucleic acid;

3) using one of said detection probes A, a mixture of plurality of thedetection probe A, one of said detection probes B and/or a mixture of aplurality of said detection probes B, hybridize with said biotin labelednucleic acid, and obtain hybridization product;

4) ligate said hybridization product using a DNA ligase, to obtain thesequencing target sequence;

5) amplify and sequence the target sequence by Barcode specific primerand Common primer to obtain sequencing result of the targetamplification product; the 20 bases of 3′ end of barcode primer andcommon primer is complementary to universal probe P1 or P2; said Barcodespecific primers containing Barcode sequence XXXXXX sample which is arandom sequence of A, T, C and G, determining different samples based ondifferent combinations;

6) analyzing the sequencing results to determine whether the test samplecontaining the mutation site or to determine the genotype of themutation site.

Embodiment 2: A method according to Embodiment 1, comprising: in step1), said detection probes are plurality of detection probes A fordetecting the SNP and plurality of detection probes B for detectingdeletion or insertion mutation sites; each probe are mixed in equimolarratio; in step 2), said plurality of said pre-amplification primerequimolar mixture of plurality of said pre-amplification primer pairs;said test sample is one or more, each barcode specific primer iscorresponding to one test sample; in Step 3), said mixture of pluralityof detection probes A is plurality of the detection probes in equimolarratio; said mixture of a plurality of the detection probe B is pluralityof the detection probe B in equimolar ratio.

Embodiment 3: The method according to Embodiment 1 or 2, comprising: instep 2), after pre-amplification, further comprising: adding exonucleaseI and alkaline phosphatase to said pre-amplification product to removepre-amplification primer and dNTP in the system, obtaining biotinlabeled nucleic acid.

Embodiment 4: The method according to any one of Embodiment s 1-3,comprising: In step 3), during hybridization, magnetic beads adsorb andcapture the hybridization product.

Embodiment 5: The method according to any one of Embodiment s 1-4,comprising: In step 5), the sequencing using second-generationsequencing instruments.

Embodiment 6: The method according to any one of Embodiment s 1-5,further comprising: said detection probes are 2 detection probe A fordetecting SNP and 3 detection probe B for detecting deletion orinsertion; said SNP sites are 1494C>T and IVS7-2A>G; said deletion orinsertion mutation site is 235delC, 176DEL16 and 299delAT; saiddetection probe A corresponding to 1494C>T comprising probe ASO-A shownin SEQ ID No. 21, probe ASO-A shown in SEQ ID No. 22, probe ASO-A shownin SEQ ID No. 23, probe ASO-A shown in SEQ ID No. 24, and probe LSO-Ashown in SEQ ID No. 25; said detection probe A corresponding toIVS7-2A>G comprising probe ASO-A shown in SEQ ID No. 7, probe ASO-Ashown in SEQ ID No. 8, probe ASO-A shown in SEQ ID No. 9, probe ASO-Ashown in SEQ ID No. 10, and probe LSO-A shown in SEQ ID No. 11; saiddetection probe A corresponding to 235delC comprising ASO-B probe shownin SEQ ID No. 15, ASO-A probe shown in SEQ ID No. 16, and LSO-B probeshown in SEQ ID No. 17; said detection probe A corresponding to 176DEL16comprising ASO-B probe shown in SEQ ID No. 12, ASO-A probe shown in SEQID No. 13, and LSO-B probe shown in SEQ ID No. 14; said detection probeA corresponding to 299delAT comprising ASO-B probe shown in SEQ ID No.18, ASO-A probe shown in SEQ ID No. 19, and LSO-B probe shown in SEQ IDNo. 20; Said pre-amplification primers comprising primer pairs 1 foramplifying 176del16, 299delAT and 235delC, primer pairs 2 for amplifying1494C>T, and primer pairs 3 for amplifying IVS7-2A>G; Said primer pairs1 comprising single strand DNA shown in SEQ ID No. 28 and single strandDNA shown in SEQ ID No. 29; Said primer pairs 2 comprising single strandDNA shown in SEQ ID No. 26 and single strand DNA shown in SEQ ID No. 27;Said primer pairs 3 comprising single strand DNA shown in SEQ ID No. 30and single strand DNA shown in SEQ ID No. 31.

Embodiment 7: A kit for detecting mutation sites comprising detectionprobe A for detecting 1494C>T, detection probe A for detectingIVS7-2A>G, detection probe B for detecting 235delC, detection probe Bfor detecting 176DEL16, detection probe B for detecting 299delAT,pre-amplification primer pairs 1 for amplifying 176del16, 299delAT and235delC, pre-amplification primer pairs 1 for amplifying 1494C>T,pre-amplification primer pairs 2 for amplifying, and pre-amplificationprimer pairs 3 for amplifying IVS7-2A>G in Embodiment 6; said mutationsites are 1494C>T, IVS7-2A>G, 235delC, 176DEL16 and 299delAT.

EXAMPLES

All the methods used in the following examples are common unlessotherwise specified. All the materials and reagents used in thefollowing examples are commercially available unless otherwisespecified. The present disclosure is illustrated by but not limited tothe following examples. The quantitative tests of the following examplesof were performed as triplicate experiments, and the results wereaveraged. FIG. 1 is an example showing a detection method of the presentdisclosure.

Example 1 Detection of Five Deafness Mutation Sites (SNP) Based onHigh-Throughput Sequencing Technology in Mixed Plasmid Samples

In this example, multiple deafness sites were detected and plasmids fordetecting the deafness sites are constructed by CapitalBio Corporation.Sequences of the plasmids were confirmed by sequencing validation. Theinformation of the plasmids is shown in Table 1, and the mutationsinclude SNP, deletion, and/or insertion.

TABLE 1 Information of the plasmids for deafness sites. Plasmid Siteinformation corresponding to mutations pGEMT-299WT 235WT 

 176WT 

 299WT pCMV-235delC 235delC 

 176WT 

 299WT pCMV-176del16 235WT 

 176del16 

 299WT pCMV-299delAT 235WT 

 176WT 

 299delAT pGEMT-IVSA IVS7-2WT pCMV-IVSG IVS7-2A > G pGEMT-1555WT 1494WTpCMV-1494C > T 1494C > T NOTE: WT means wild type, del means deletion,and > means SNP mutation.

Construction of Plasmid pGEMT-299WT:

human genomic DNA was amplified using primers XPMS0299F/XPMS0299R toobtain gene fragment 299WT containing the 235, 176, 299 sites. The 299WTfragment was then cloned into the vector pGEMT-easy.

Point mutations at site 235, 176, or 299 of the fragment 299WT wereintroduced, to obtain 235delC (in which the 235 C in the GJB2 gene wasdeleted, Reference Sequence GenBank No.: KF638275.1, submitted Nov. 30,2013), 176del16 (in which the 16 residues of sites 176-191 in the GJB2gene were deleted, Reference Sequence GenBank No.: KF638275.1, submittedNov. 30, 2013), 299delAT (in which the 299-300 AT residues in the GJB2gene were deleted, Reference Sequence GenBank No.: KF638275.1, submittedNov. 30, 2013). Then the fragments were cloned into plasmid PCMV toobtain plasmids pCMV-235delC, pCMV-176del16, and pCMV-299delAT.

Construction of Plasmid pGEMT-IVSA:

human genomic DNA was amplified using primers XPMS0919F/XPMS0919R toobtain gene fragment IVSA containing the IVS7-2 site (the IVS7-2 site ofthe SLC26A4 gene, Reference Sequence GenBank No.: NG_008489, submittedJul. 25, 2013). The fragment was then cloned into the vector pGEMT-easy.

Point mutations at IVSA sites of the IVS7-2 fragment were introduced, toobtain the IVS7-2A>G fragment (in which the A>G mutation occurred at theIVS7-2 site of the SLC26A4 gene, Reference Sequence GenBank No.:NG_008489, submitted Jul. 25, 2013). Then the fragments were cloned intoplasmid PCMV by Nae I to obtain plasmid pCMV-IVSG.

Construction of Plasmid pGEMT-1555WT:

human genomic DNA was amplified using primers XPMS1555F/XPMS1555FR toget the gene fragment 1494WT containing the 1494 site (the 1494 site ofmitochondrial gene 12SrRNA, Reference Sequence GenBank No.: J01415.2,submitted Jul. 17, 2013). The fragment was then cloned into the vectorpGEMT-easy.

Point mutation at the 1494 site was introduced into the sequence offragment 1494WT, to obtain the 1494C>T fragment (in which the C>Tmutation occurred at the 1494 site in mitochondrial 12SrRNA gene,Reference Sequence GenBank No.: J01415.2, submitted Jul. 17, 2013). Thenthe fragments were cloned into plasmid PCMV by Nae I to obtain plasmidpCMV-1494C>T.

TABLE 2 Primers used for plasmid construction. Primer SequencesXPMS0299F CCAGACTCAGAGAAGTCTCCC (SEQ ID No. 1) XPMS0299RATGCTAGCGACTGAGCCTTGA (SEQ ID No. 2) XPMS0919FCGTGTAGCAGCAGGAAGTAT (SEQ ID No. 3) XPMS0919RAAGAGGAACACCACACTCAC (SEQ ID No. 4) XPMS1555FTGGCTAAGGTTGTCTGGTAG (SEQ ID No. 5) XPMS1555RCCCTGATGAAGGCTACAAAG (SEQ ID No. 6)

(I) Probe and Pre-Amplification Primer Design

(1) Test Sample Preparation

After quantification of the plasmids in Table 1, the plasmids were mixedas follows:

Experimental Group I:

The pGEMT-299WT, pGEMT-IVSA, and pGEMT-1555WT plasmids were mixed atequal copy numbers, e.g., 5×10³ copies. In other words, the copy numberof each of the 235WT, 176WT, 299WT, IVS7-2WT, and 1494WT plasmids was5×10³. Therefore, the five plasmids were present in the mixture at equalconcentrations. The mixed plasmids were then diluted to obtain six testsamples in which the copy number of each plasmid was 5×10³, 10³, 5×10²,10², 5×10¹, and 10¹, respectively.

Experimental Group II:

5×10³ copies of each of plasmids pGEMT-299WT, pGEMT-IVSA, pGEMT-1555WT,pCMV-235delC, pCMV-299delAT, pCMV-176del16, pCMV-1494C>T, and pCMV-IVSGwere mixed. The mixed plasmids were then diluted to obtain six testsamples in which the copy number of each plasmid was 5×10³, 10³, 5×10²,10², 5×10¹, and 10¹, respectively.

In Experimental Group II, according to the site information, in themixed plasmids, 50% of the SLC26A4 plasmids were the mutated pCMV-IVSGplasmids (because pGEMT-IVSA and pCMV-IVSG had equal copy numbers), withdilution gradients of 5×10³, 10³, 5×10², 10², 5×10¹, and 10¹. 50% of the12SrRNA plasmids were the mutated pCMV-1494C>T plasmids (becausepGEMT-1555WT and pCMV-1494C>T had equal copy numbers), with dilutiongradients of 5×10³, 10³, 5×10², 10², 5×10¹, and 10¹. Because thepGEMT-299WT, pCMV-235delC, pCMV-299delAT, and pCMV-176del16 plasmids hadequal copy numbers, each of these plasmids represented 25% of the GJB2plasmids, with dilution gradients of each mutation at 10⁴, 2×10³, 4×10²,2×10², and 4×10¹.

Experimental Group III:

5×10³ copies of each of plasmids pCMV-235delC, pCMV-299delAT,pCMV-176de116, pCMV-1494C>T, and pCMV-IVSG were mixed. The mixedplasmids were then diluted to obtain six test samples in which the copynumber of each plasmid was 5×10³, 10³, 5×10², 10², 5×10¹, and 10¹,respectively.

In Experimental Group III, according to the site information, in themixed plasmids, 100% of the SLC26A4 plasmids were the mutated pCMV-IVSGplasmids, with dilution gradients of 5×10³, 10³, 5×10², 10², 5×10¹, and10¹. 100% of the 12SrRNA plasmids were the mutated pCMV-1494C>Tplasmids, with dilution gradients of 5×10³, 10³, 5×10², 10², 5×10¹, and10¹. Because the pCMV-235delC, pCMV-299delAT, and pCMV-176del16 plasmidshad equal copy numbers, each of these plasmids represented 33% of theGJB2 plasmids, with dilution gradients of each mutation at 7.5×10³,3×10³, 1.5×10³, 3×10², 1.5×10², and 3×10¹.

(2) Probe and Pre-Amplification Primer Design

1) Probe Design

Principle:

Detection probes can be designed according to 40 bp nucleotides flankingthe mutation site, for example, an SNP or an insertion and/or deletionmutation. Detection Probes A are used for detecting SNP sites, andDetection Probes B are used for detecting insertion and/or deletionsites. Each detection probe is between about 13 bp and about 25 bp.

Detection Probes A for detecting SNP sites can include the upstreamgenotyping probe ASO-A and the downstream site-specific probe LSO-A, andASO-A and LSO-A probes have the same extending direction.

In one aspect, the 3′ end of the ASO-A probe is identical orcomplimentary to the target sequence in which the wild-type or mutantresidue at the SNP site is present. The 3′ terminal nucleotide of theASO-A probe is identical or complimentary to the wild-type or mutantresidue of the SNP site. In another aspect, the 5′ end of the ASO-Aprobe is separated from the target sequence in which the wild-type ormutant residue at the SNP site is present. In some aspects, the ASO-Aprobe comprises a 5′ end sequence that is not identical or complimentaryto the target sequence, and the 5′ end sequence does not hybridize tothe target sequence or a complementary sequence thereof. In someaspects, the 5′ end sequence comprises a common probe sequence P1.

An ASO-A probe can be provided to correspond to each wildtype or mutantresidue of a SNP site. For example, an ASO-A probe can be provided tocorrespond to 1494C of the 12SrRNA gene, and another ASO-A probe can beprovided to correspond to 1494T of the 12SrRNA gene.

The 5′ end of the LSO-A probe is identical or complementary to thetarget sequence. The 5′ terminal nucleotide of the LSO-A, with aterminal nucleotide phosphorylation, is identical or complementary tothe first nucleotide, of the target sequence, downstream of the SNP sitein the extending direction of the probe. In one aspect, the 3′ end ofthe LSO-A probe is separated from the target sequence in which the SNPsite is present. In some aspects, the LSO-A probe comprises a 3′ endsequence that is not identical or complimentary to the target sequence,and the 3′ end sequence does not hybridize to the target sequence or acomplementary sequence thereof. In some aspects, the 3′ end sequence ofthe LSO-A comprises a universal probe sequence P2, for example, the22nd-42nd nucleotides from the 5′ end of SEQ ID NO: 11.

Detection Probes B for detecting insertion/deletion mutation can includean upstream genotyping probe ASO-B(1) for detecting an insertion and/ordeletion mutation, an upstream genotyping probe ASO-B(2) for detectingthe wild-type sequence, and a downstream site-specific probe LSO-B. Allthree probes have the same extending direction.

The upstream genotyping probe ASO-B for detecting the insertion and/ordeletion mutation can include the ASO-B probe for detecting aninsertion, and the ASO-B probe for detecting a deletion.

In one aspect, the 3′ end of the ASO-B probe for detecting an insertionis identical or complimentary to the target sequence in which theinsertion sequence is present. The 3′ terminal nucleotide of the ASO-Bprobe is identical or complimentary to the terminal nucleotide of theinsertion sequence.

In one aspect, the 3′ end of the ASO-B probe for detecting a deletion isidentical or complimentary to the target sequence in which the deletionis present. The 3′ terminal nucleotide of the ASO-B probe is identicalor complimentary to the first nucleotide immediately upstream to thedeletion site.

Target sequence of insertion/deletion mutation sites is the wild-typetarget sequence with an insertion and/or deletion before the n^(th)base.

The 5′ end of the downstream site-specific probe LSO-B is identical orcomplementary to the target sequence of the wild-type site. The 5′terminal nucleotide is identical or complementary to the n^(th)nucleotide of the wild-type site sequence in the probe extendingdirection.

In one aspect, the 3′ end of the LSO-B probe is separated from thetarget sequence. In some aspects, the LSO-B probe comprises a 3′ endsequence that is not identical or complimentary to the target sequence,and the 3′ end sequence does not hybridize to the target sequence or acomplementary sequence thereof. In some aspects, the 3′ end sequence ofthe LSO-B comprises a universal probe sequence P2.

In some aspects, each upstream genotyping probe ASO and thecorresponding downstream site-specific probe LSO of a mutation site canbe spliced together without any overlapping nucleotide(s) or gap. Thespliced sequence can provide the reverse complement sequence of thetarget sequence, either wild-type or mutant.

For example, Detection Probes A can include four upstream genotypingprobes (ASO) and one downstream site-specific probe (LSO), as shown inFIG. 1. The ASO and LSO probes have the same extension direction, andcan be spliced together without any overlap or gap, to obtain thereverse complement sequence of the target sequence.

In FIG. 1, four ASO-A probes can be used to detect a SNP. The 3′terminal nucleotide of the four ASO-A probes are A, T, C, and G,respectively; the 5′ end of the ASO-A probes separate from the targetsequence and comprise a universal probe P1 (for example, the1^(st)-20^(th) nucleotides of the 5′ end of SEQ ID NO: 7).

In some examples, three of the four ASO-A probes may be used, and two ofthe ASO-A probes can comprise a 3′ terminal nucleotide that correspondsto the wild-type residue and the mutant residue of the SNP,respectively. The 3′ end of the other ASO-A probe is different from thewild-type and the mutant residue of the SNP. The 5′ ends of the ASO-Aprobes are separate from the target sequence and can comprise theuniversal probe P1 (for example, the 1^(st)-20^(th) nucleotides of the5′ end of SEQ ID NO: 7).

Alternatively, two of the four ASO-A probes may be used, each comprisinga 3′ terminal nucleotide that corresponds to the wild-type residue andthe mutant residue of the SNP, respectively. The 5′ ends of the ASO-Aprobes are separate from the target sequence and can comprise theuniversal probe P1 (for example, the 1^(st)-20^(th) nucleotides of the5′ end of SEQ ID NO: 7).

When detecting one particular SNP (i.e., one residue at the SNP site),one of the four ASO-A probes can be used, and the 3′ end base of theASO-A probe is identical or complementary to the SNP residue to bedetected. The 5′ end of the ASO-A probe is separate from the targetsequence and can comprise the universal probe P1 (for example, the1^(st)-20^(th) nucleotides of the 5′ end of SEQ ID NO: 7).

The 5′ end of the LSO probe with a terminal nucleotide phosphorylationis complementary to the target sequence, and the 5′ terminal base of theLSO probe is complementary to the next base of the target sequencedownstream of the SNP site in the probe extending direction. The 3′ endof the LSO probe is separate from the target sequence and can comprisethe universal probe P2 (for example, the 22^(nd)-42^(nd) nucleotidesfrom the 5′ end of SEQ ID NO: 11).

Detection Probes B can include the upstream genotyping probe fordetecting the mutant sequence (ASO), the upstream genotyping probe fordetecting the wild-type sequence (ASO), and the downstream site-specificprobe (LSO). The probes have the same extension direction. In oneaspect, the upstream genotyping probe and the downstream site-specificprobe for detecting the mutant sequence can be spliced together withoutoverlap or gap, to obtain the reverse complement sequence of the targetsequence. In another aspect, the upstream genotyping probe and thedownstream site-specific probe for detecting the wild-type sequence canbe spliced together without overlap or gap, to obtain the reversecomplement sequence of the wild-type target sequence. The mutant targetsequence, in one aspect, is the wild-type target sequence with aninsertion or deletion before the n^(th) base.

According to the above design principles, detection probes were designedfor the about 40 bp flanking sequences of the five deafness associatedmutation sites (235delC, 176del16, 299delAT, IVS7-2A>G, 1494C>T). Inorder to ensure the sensitivity and specificity of the probes, thehybridizing region of the probe and the template is 13 nt for the 1494site in the 12SrRNA gene and with a mutation GcCCG to GgCCG.

TABLE 3 Probe sequences. Probe Type (name / type / mutation type /Probes Sequences mutation bases) IVSUG17ACACGACGCTCTTCCGATCTAGTAGCAATTATCGTCT (SEQ ID No. 7) IVSUG17/ASO/snp/TIVSUC17 ACACGACGCTCTTCCGATCTAGTAGCAATTATCGTCA (SEQ ID No. 8)IVSUG17/ASO/snp/A IVSUA17ACACGACGCTCTTCCGATCTAGTAGCAATTATCGTCG (SEQ ID No. 9) IVSUG17/ASO/snp/GIVSUT17 ACACGACGCTCTTCCGATCTAGTAGCAATTATCGTCC (SEQ ID No. 10)IVSUG17/ASO/snp/C IVSD3GAAATAAAACAAAAGATGTTAGATCGGAAGAGCACACGTCT (SEQ ID No. IVSUG17/LSO 11)176WU-2 ACACGACGCTCTTCCGATCTCCAGGCTGCAAGAACGTGTG (SEQ ID No. 12)176/ASO/wild-type(WT) 176MU-2ACACGACGCTCTTCCGATCTTGCAACACCCTGCAGCCAG (SEQ ID No. 13)176/ASO/deletion(MT) 176DPCTACGATCACTACTTCCCCAGATCGGAAGAGCACACGTCT (SEQ ID No. 14) 176/LSO 235WUACACGACGCTCTTCCGATCTACATCCGGCTATGGGCC (SEQ ID No. 15)235/ASO/ wild-type (WT) 235MUACACGACGCTCTTCCGATCTCACATCCGGCTATGGGC (SEQ ID No. 16)235/ASO/deletion(MT) 235DPCTGCAGCTGATCTTCGTGTCGATCGGAAGAGCACACGTCT (SEQ ID No. 17) 235/LSO 299WUACACGACGCTCTTCCGATCTTGGCCTACCGGAGACAT (SEQ ID No. 18)235/ASO/ wild-type (WT) 299MUACACGACGCTCTTCCGATCTCGTGGCCTACCGGAGAC (SEQ ID No. 19)299/ASO/deletion(MT) 299DPGAGAAGAAGAGGAAGTTCATGATCGGAAGAGCACACGTCT (SEQ ID No. 299/LSO 20)1494UG13 ACACGACGCTCTTCCGATCTACCGgCCGTCACG (SEQ ID No. 21)1494/ASO/snp/G 1494UC13ACACGACGCTCTTCCGATCTACCGgCCGTCACC (SEQ ID No. 22) 1494/ASO/snp/C1494UA13 ACACGACGCTCTTCCGATCTACCGgCCGTCACA (SEQ ID No. 23)1494/ASO/snp/A 1494UT13ACACGACGCTCTTCCGATCTACCGgCCGTCACT (SEQ ID No. 24) 1494/ASO/snp/T 1494DPCTCCTCAAGTATACTTCAAAGATCGGAAGAGCACACGTCT (SEQ ID No. 25) 1494/L50

2) Pre-Amplification Primer Design

Design Principle: Based on the nucleotide sequences to be detected,primers are designed to amplify the target sequence containing themutation site. The primers are then used for sample pre-amplificationand biotin labeling.

Pre-amplification primers were designed according to the 5 deafnessmutations (IVS7-2A>G, 176del16 (16 bp deletion at the 176 site),299delAT, 235delC, and 1494C>T).

TABLE 4 Pre-amplification primers sequences. Primers Sequences 1555F1AGTGCTTAGTTGAACAGGGCC (SEQ ID No. 26) 1555R1-BBiotin-GGTTCGTCCAAGTGCACTTTC (SEQ ID No. 27) GJB234F1AAAGGAGGTGTGGGGAGATGA (SEQ ID No. 28) GJB234R1-BBiotin-CTGGGTTTTGATCTCCTCGATG (SEQ ID No. 29) IVS7-2F1-BBiotin-tcaCCATTGTCGTCTGTATGGCA (SEQ ID No. 30) IVS7-2R1CCAGGTTGGCTCCATATGAAatg (SEQ ID No. 31)

The amplification products of GJB234F1/GJB234R1-B contain sequencescomprising 176del16, 299delAT, and/or 235delC. The amplificationproducts of 1555F1/1555R1-B contain sequences comprising the 1494C>Tsite. The amplification products of IVS7-2F1-B/IVS7-2R1 containsequences comprising the IVS7-2A>G site.

The equimolar mix of the above six primers is named Multi-p3.

(II) Pre-Amplification

Pre-amplification was conducted using the above-mentioned Multi-p3 ofthe six samples in each group of Experimental Groups 1-3.

The pre-amplification system volume was 10 μL, containing 1 μL DNAtemplate, 1 μL 1×PCR buffer, 0.5 U DNA polymerase, 1 μL biotin labeledmixed primers Multi-p3 (50 nM each for primer), 200 μM dNTP, 4.5 mMMgCl₂, with water added to bring up the total volume to 10 μL.

PCR condition: first, 95 degrees (Celsius) denaturation for 5 min, then95 degrees (Celsius) denaturation for 20 s, 60 degrees (Celsius)annealing for 4 min; 20 Cycles. Biotin label was added in thepre-amplification process. After pre-amplification, final concentration1 U/μL Exonuclease I (Exol) and final concentration 0.1 U/μL alkalinephosphatase (AP) were added to the amplified products to remove primersand dNTPs in the pre-amplification system. After 37 degrees (Celsius)reaction for 3 h, 6 biotin-labeled nucleic acid samples in ExperimentalGroup 1, 6 biotin-labeled nucleic acid samples in Experimental Group 2,and 6 biotin-labeled nucleic acid samples in Experimental Group 3 wereobtained.

(III) Hybridization

The 19 probes in Table 3 were mixed in equal molar ratio to get thedetection probes set.

Each biotin-labeled nucleic acid sample obtained in (II) above washybridized with the detection probes set (each probe 100 at fmol), thenthe hybridization products were captured and absorbed by magnetic beads.

For example, each of the biotin-labeled nucleic acid sample (8 μL) andthe detection probes set 2 μl (concentration of each probe—50 pM) wereadded into 20 μL hybridization buffer (2×) (100 mM Tris-HCl, 500 mMNaCl, 1 mM EDTA, 0.2% tween80, pH=7.6). The mixture was then subjectedto high temperature denaturation, followed by low temperature annealing(95 degrees (Celsius) for 5 min, then 45 degrees (Celsius) for 10 minannealing), so that the probe ASO and LSO fully hybridized with flanksequences of the detection sites. Magnetic beads were then added,followed by 45 degrees (Celsius) incubation for 2 h. 1× washing buffer(50 mM Tris-HCl, 100 mM NaCl, 0.5 mM EDTA, 0.1% tween80, pH=7.6) and Taqligation buffer (NEB) were used to wash the magnetic beads to remove thenon-hybridized probes, obtaining 6 hybridization products inExperimental Group 1, 6 hybridization products in Experimental Group 2,and 6 hybridization products in Experimental Group 3.

(IV) Ligation

DNA ligase was added to the 6 hybridization products in ExperimentalGroup 1, the 6 hybridization products in Experimental Group 2, and the 6hybridization products in Experimental Group 3, and ligation reactionswere performed. After ligation of perfectly matched probes, the ligationreaction buffer was removed, and the beads were re-suspended by ddH₂O.Then the reaction was incubated at 95 degrees (Celsius) for 5 min tomelt the hybridized probes and obtain 6 ligated sequences inExperimental Group 1, 6 ligated sequences in Experimental Group 2 and 6ligated sequences in Experimental Group 3.

(V) Amplification and Sequencing

Barcode primer: (SEQ ID NO: 32)CAAGCAGAAGACGGCATACGAGATAXXXXXXGTGACTGGAGT TCAGACGTGTGCTCTTCCGATCT.XXXXXX is the barcode sequence to differentiate the samples, and the 6bases can be random combinations of A, T, C, and G, to distinguish eachsample.

Common primer: (SEQ ID NO: 33)AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTAC ACGACGCTCTTCCGATCT.

The 20 bases of the 3′ end of the barcode primer and the common primerare complementary sequences to P1 and P2, respectively. Using thebarcode primer and the common primer, the 6 ligated sequences inExperimental Group 1, the 6 ligated sequences in Experimental Group 2,and the 6 ligated sequences in Experimental Group 3 were amplified byPCR. The amplified products were then extracted and sequencing wasperformed using second generation high-throughput sequencinginstruments.

(VI) Data Analysis

Sequence alignment was conducted between the sequencing results and thereverse complementary sequences of each mutant or wild-type targetsequence. The number of sequencing results that matched the targetsequence was counted and used to calculate the proportion of the mutantsin each sample.

The proportion of the mutants is the ratio of the mutation sites to thetotal sites, and the total site is the total of the mutation sites andwild-type sites. The proportion of the mutation was used to judgegenotype information of the sites, using statistical analysis andsetting appropriate threshold, then judging genotype information of thelocus by the threshold.

In this example, if the mutation site ratio is greater than 90%, thetest sample contains or likely contains the mutation site, or thegenotype of test sample is homozygous of the mutation site or acandidate of homozygous mutation site. If the mutation site ratio is20-90%, the test sample contains or likely contains the mutation site,or the genotype of test sample is a heterozygous mutation site or acandidate of heterozygous mutation site. If the mutation site ratio isless than 20%, the test sample does not contain or likely does notcontain the mutation site, or the genotype of test sample is wild-typeor a candidate for wild-type sample.

Experimental results showed that using the methods disclosed herein, forplasmid group I (in which the 1494 site, IVS7-2 site, 176 site, 235site, and 299 site were all wild-type), the sensibility was 5×10¹ copy(FIG. 2); for plasmid group II (in which the 1494 sites were all mutant,50% of the IVS7-2 sites were mutant, 25% of the 176 sites, 235 sites, or299 sites were mutant), the sensibility of the 1494 sites and the IVS7-2sites was 10¹ copy, the sensibility of the 176 sites, 235 sites, or 299sites was 4×10¹ copy (FIG. 3); for plasmid group III (the IVS7-2 sitesand the 1494 sites were all mutant, 33.3% of the 235 sites, 176 sites,and 299 sites were mutant), the sensibility of the 1494 sites and theIVS7-2 sites was 5×10¹ copy, the sensibility of the 176 sites, the 235sites, or the 299 sites was 3×10¹ copy (FIG. 4).

Example 2 Detection of SNP Based on High-Throughput SequencingTechnology in Human Genomic DNA Samples

Probe and Pre-Amplification Primer Design

1. Test Sample Preparation

Deafness patients' blood genomic DNA (verified homozygous mutant genomicDNA at the 235 residue of the GJB2 gene) and normal human blood genomicDNA (wild-type genomic DNA) were used, and each sample was repeated forthree times. The concentration of nucleic acid was 10 ng/μL.

2. Probe and Pre-Amplification Primer Design

1) Probe Design

The principle of design is the same as in Example 1. The sequences ofthe probes are listed in Table 5.

TABLE 5 Probes. 176WU-2 ACACGACGCTCTTCCGATCTCCAGGCTGCAAGAACGTGTG176/ASO/wild-type(WT) (SEQ ID No.: 12) 176MU-2ACACGACGCTCTTCCGATCTTGCAACACCCTGCAGCCAG 176/ASO/ Deletion Type(SEQ ID No.: 13) (MT) 176DP CTACGATCACTACTTCCCCAGATCGGAAGAGCACACGTCT176/LSO (SEQ ID NO: 14) 235WU ACACGACGCTCTTCCGATCTACATCCGGCTATGGGCC (SEQ235/ASO/ WT ID NO: 15) 235MU ACACGACGCTCTTCCGATCTCACATCCGGCTATGGGC (SEQ235/ASO/ MT ID NO: 16) 235DP CTGCAGCTGATCTTCGTGTCGATCGGAAGAGCACACGTCT235/LSO (SEQ ID NO: 17) 299WU ACACGACGCTCTTCCGATCTTGGCCTACCGGAGACAT (SEQ235/ASO/ WT ID NO: 18) 299MU ACACGACGCTCTTCCGATCTCGTGGCCTACCGGAGAC (SEQ299/ASO/ MT ID NO: 19) 299DP GAGAAGAAGAGGAAGTTCATGATCGGAAGAGCACACGTCT299/LSO (SEQ ID NO: 20)

2). Probe and Pre-Amplification Primer Design

The principle of design is the same as in Example 1. The primers usedwere GJB234F1/GJB234R1-B.

Pre-amplification, hybridization, ligation, amplification, and dataanalysis were performed substantially the same as in Example 1.

Results are shown in FIG. 5. The genomic DNA of deafness patient washomozygous mutation at position 235, wild-type at position 176, andwild-type at position 299. The results were consistent with the samplesetup.

The invention claimed is:
 1. A probe set for analyzing a genetic locusof a target polynucleotide sequence, comprising: one or more firstprobes comprising: (1) a first hybridization sequence that specificallybinds to the target polynucleotide sequence upstream of or including thegenetic locus, and (2) a first primer sequence upstream of the firsthybridization sequence, wherein the first primer sequence does not bindto the target polynucleotide sequence; and one or more second probescomprising: (i) a second hybridization sequence that specifically bindsto the target polynucleotide sequence downstream of or starting from thegenetic locus, and (ii) a second primer sequence downstream of thesecond hybridization sequence, wherein the second primer sequence doesnot bind to the target polynucleotide sequence, wherein: the extensiondirections of the first and second probes are the same; the first probeis upstream of the second probe; and the first and second probes areadjacent and, when coupled, form a sequence comprising the geneticlocus, and wherein: a) the genetic locus comprises an insertion at then^(th) residue of a wild-type target polynucleotide sequence; and theone or more first probes comprise at least two first probes, one ofwhich specifically binds to the wild-type target polynucleotide sequenceuntil and excluding the n^(th) residue, while the other specificallybinds to the target polynucleotide sequence including and until the lastresidue of the inserted sequence, and the second hybridization sequenceof the one or more second probes specifically binds to the targetpolynucleotide sequence downstream of the insertion, and the 5′ terminalnucleotide of the one or more second probes is complementary to n^(th)residue; or the one or more second probes comprise at least two secondprobes, one of which specifically binds to the wild-type targetpolynucleotide sequence starting from the n^(th) residue, while theother specifically binds to the target polynucleotide sequence includingand from the first residue of the inserted sequence, and the firsthybridization sequence of the one or more first probes specificallybinds to the target polynucleotide sequence upstream of the insertion,and the 3′ terminal nucleotide of the one or more first probes iscomplementary to the residue immediately upstream of n^(th) residue inthe wild-type target polynucleotide; or b) the genetic locus comprises adeletion at the n^(th) residue of a wild-type target polynucleotidesequence; and the one or more first probes comprise at least two firstprobes, one of which specifically binds to the wild-type targetpolynucleotide sequence until and excluding the n^(th) residue, whilethe other specifically binds to the target polynucleotide sequence untilthe first residue immediately upstream of the deleted sequence, and thesecond hybridization sequence of the one or more second probesspecifically binds to the target polynucleotide sequence downstream ofthe deletion, and the 5′ terminal nucleotide of the one or more secondprobes is complementary to n^(th) residue; or the one or more secondprobes comprise at least two second probes, one of which specificallybinds to the wild-type target polynucleotide sequence starting from then^(th) residue, while the other specifically binds to the targetpolynucleotide sequence including and from the first residue of thedeleted sequence, and the first hybridization sequence of the one ormore first probes specifically binds to the target polynucleotidesequence upstream of the deletion, and the 5′ terminal nucleotide of theone or more first probes is complementary to the residue immediatelyupstream of n^(th) residue in the wild-type target polynucleotide. 2.The probe set of claim 1, wherein: the genetic locus comprises an SNP ora point mutation; and the first hybridization sequence of the one ormore first probes specifically binds to the target polynucleotidesequence including the SNP or point mutation, and the 3′ terminalnucleotide of the one or more first probes is complementary to thenucleotide at the SNP or point mutation locus, and the secondhybridization sequence of the one or more second probes specificallybinds to the target polynucleotide sequence downstream of the SNP orpoint mutation, and the 5′ terminal nucleotide of the one or more secondprobes is complementary to the nucleotide immediately downstream of theSNP or point mutant locus; or the first hybridization sequence of theone or more first probes specifically binds to the target polynucleotidesequence upstream of the SNP or point mutation, and the 3′ terminalnucleotide of the one or more first probes is complementary to thenucleotide immediately upstream of the SNP or point mutation locus, andthe second hybridization sequence of the one or more second probesspecifically binds to the target polynucleotide sequence starting fromthe SNP or point mutation, and the 5′ terminal nucleotide of the one ormore second probes is complementary to the nucleotide at the SNP orpoint mutation locus.
 3. The probe set of claim 2, wherein the firstprimer sequence of the one or more first probes is unique for theresidue at the SNP or mutant locus.
 4. The probe set of claim 1, whereinthe 5′ terminus of the one or more second probes is phosphorylated. 5.The probe set of claim 1, wherein the first primer sequence of the oneor more first probes is a universal primer sequence.
 6. The probe set ofclaim 1, wherein the second primer sequence of the one or more secondprobes is a universal primer sequence.
 7. The probe set of claim 1,wherein: the genetic locus comprises an insertion at the n^(th) residueof a wild-type target polynucleotide sequence; and the one or more firstprobes comprise at least two first probes, one of which specificallybinds to the wild-type target polynucleotide sequence until andexcluding the n^(th) residue, while the other specifically binds to thetarget polynucleotide sequence including and until the last residue ofthe inserted sequence, and the second hybridization sequence of the oneor more second probes specifically binds to the target polynucleotidesequence downstream of the insertion, and the 5′ terminal nucleotide ofthe one or more second probes is complementary to n^(th) residue; or theone or more second probes comprise at least two second probes, one ofwhich specifically binds to the wild-type target polynucleotide sequencestarting from the n^(th) residue, while the other specifically binds tothe target polynucleotide sequence including and from the first residueof the inserted sequence, and the first hybridization sequence of theone or more first probes specifically binds to the target polynucleotidesequence upstream of the insertion, and the 3′ terminal nucleotide ofthe one or more first probes is complementary to the residue immediatelyupstream of n^(th) residue in the wild-type target polynucleotide. 8.The probe set of claim 7, wherein the first primer sequence of the oneor more first probes is a universal primer sequence.
 9. The probe set ofclaim 7, wherein the two first probes comprise different first primersequences.
 10. The probe set of claim 7, wherein the 5′ terminus of theone or more second probes is phosphorylated.
 11. The probe set of claim7, wherein the second primer sequence of the one or more second probesis a universal primer sequence.
 12. The probe set of claim 1, wherein:the genetic locus comprises a deletion at the n^(th) residue of awild-type target polynucleotide sequence; the one or more first probescomprise at least two first probes, one of which specifically binds tothe wild-type target polynucleotide sequence until and excluding then^(th) residue, while the other specifically binds to the targetpolynucleotide sequence until the first residue immediately upstream ofthe deleted sequence, and the second hybridization sequence of the oneor more second probes specifically binds to the target polynucleotidesequence downstream of the deletion, and the 5′ terminal nucleotide ofthe one or more second probes is complementary to n^(th) residue; or theone or more second probes comprise at least two second probes, one ofwhich specifically binds to the wild-type target polynucleotide sequencestarting from the n^(th) residue, while the other specifically binds tothe target polynucleotide sequence including and from the first residueof the deleted sequence, and the first hybridization sequence of the oneor more first probes specifically binds to the target polynucleotidesequence upstream of the deletion, and the 5′ terminal nucleotide of theone or more first probes is complementary to the residue immediatelyupstream of n^(th) residue in the wild-type target polynucleotide. 13.The probe set of claim 1, wherein the genetic locus is in a deafnessrelated gene.
 14. The probe set of claim 13, wherein the one or morefirst probes for 1494C>T comprises the polynucleotide sequence set forthin SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and/or SEQ ID NO: 24.15. The probe set of claim 13, wherein the one or more second probes for1494C>T comprises the polynucleotide sequence set forth in SEQ ID NO:25.
 16. The probe set of claim 13, wherein the one or more first probesfor IVS7-2A>G comprises the polynucleotide sequence set forth in SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and/or SEQ ID NO:
 10. 17. The probeset of claim 13, wherein the one or more second probes for IVS7-2A>Gcomprises the polynucleotide sequence set forth in SEQ ID NO:
 11. 18.The probe set of claim 13, wherein the one or more first probes for235delC comprises the polynucleotide sequence set forth in SEQ ID NO: 15and/or SEQ ID NO:16.
 19. The probe set of claim 13, wherein the one ormore second probes for 235delC comprises the polynucleotide sequence setforth in SEQ ID NO:
 17. 20. The probe set of claim 13, wherein the oneor more first probes for 176DEL16 comprises the polynucleotide sequenceset forth in SEQ ID NO: 12 and/or SEQ ID NO:13.
 21. The probe set ofclaim 13, wherein the one or more second probes for 176DEL16 comprisesthe polynucleotide sequence set forth in SEQ ID NO:
 14. 22. The probeset of claim 13, wherein the one or more first probes for 299delATcomprises the polynucleotide sequence set forth in SEQ ID NO: 18 and/orSEQ ID NO:19.
 23. The probe set of claim 13, wherein the one or moresecond probes for 299delAT comprises the polynucleotide sequence setforth in SEQ ID NO:
 20. 24. The probe set of claim 13, wherein thedeafness related gene is GJB2, SLC26A4, or 12SrRNA.
 25. The probe set ofclaim 24, wherein the genetic locus comprises 1494C>T, IVS7-2A>G,235delC, 176DEL16, and/or 299delAT.
 26. A kit for analyzing a geneticlocus, comprising the probe set of claim
 1. 27. The kit of claim 26,further comprising a primer pair for amplifying 176del16, 299delAT,and/or 235delC.
 28. The kit of claim 27, wherein the primer pair foramplifying 176del16, 299delAT, and/or 235delC comprises thepolynucleotide sequences set forth in SEQ ID NO: 28 and SEQ ID NO:29.29. The kit of claim 26, further comprising a primer pair for amplifying1494C>T.
 30. The kit of claim 29, wherein the primer pair for amplifying1494C>T comprises the polynucleotide sequences set forth in SEQ ID NO:26 and SEQ ID NO:
 27. 31. The kit of claim 26, further comprising aprimer pair for amplifying IVS7-2A>G.
 32. The kit of claim 31, whereinthe primer pair for amplifying IVS7-2A>G comprises the polynucleotidesequences set forth in SEQ ID NO: 30 and SEQ ID NO:
 31. 33. The kit ofclaim 26, wherein one or both of the primers of the primer pair arelabeled.
 34. The kit of claim 33, wherein the label comprises biotin.35. The kit of claim 26, further comprising a barcode specific primerand/or a common primer.
 36. The kit of claim 35, wherein the barcodespecific primer comprises the polynucleotide sequences set forth in SEQID NO: 32, and the common primer comprises the polynucleotide sequencesset forth in SEQ ID NO:
 33. 37. A composition for analyzing at least afirst genetic locus and a second genetic locus, comprising a first probeset of claim 1 for the first genetic locus, and a second probe set ofclaim 1 for the second genetic locus.
 38. The composition of claim 37,wherein the probes in the first probe set are in equal molar amount, andthe probes in the second probe set are in equal molar amount.
 39. Thecomposition of claim 37, wherein the first genetic locus comprises anSNP or point mutation, and the second genetic locus comprises a deletionand/or insertion.
 40. The composition of claim 39, wherein the SNP orpoint mutation comprises 1494C>T, and/or IVS7-2A>G, and the deletionand/or insertion comprise 235delC, 176DEL16, and/or 299delAT.
 41. Thecomposition of claim 37, wherein the first and second probe setscomprise at least two of the following probe sets: (1) a first probecomprising the polynucleotide sequences set forth in SEQ ID NO: 25, andone or more of a second probe comprising the polynucleotide sequencesset forth in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO:24; (2) a first probe comprising the polynucleotide sequences set forthin SEQ ID NO: 11, and one or more of a second probe comprising thepolynucleotide sequences set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, or SEQ ID NO: 10; (3) a first probe comprising the polynucleotidesequences set forth in SEQ ID NO: 17, and one or more of a second probecomprising the polynucleotide sequences set forth in SEQ ID NO: 15 orSEQ ID NO: 16; (4) a first probe comprising the polynucleotide sequencesset forth in SEQ ID NO: 14, and one or more of a second probe comprisingthe polynucleotide sequences set forth in SEQ ID NO: 12 or SEQ ID NO:13; and (5) a first probe comprising the polynucleotide sequences setforth in SEQ ID NO: 20, and one or more of a second probe comprising thepolynucleotide sequences set forth in SEQ ID NO: 18 or SEQ ID NO: 19.